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A systematic study on the surface-initiated polymerization of di(ethylene glycol) methyl ether methacrylate (DEGMA) by atom transfer radical polymerization (ATRP) from glass, silicon, titanium as well as tissue culture polystyrene (TCPS) is reported in an attempt to expand the known thermoresponsive poly(di(ethylene glycol) methyl ether methacrylate) (PDEGMA) cell release layers on gold to other substrates. The use of these substrate materials requires an altered immobilization chemistry to couple a bromide containing ATRP initiator to the surfaces. Using aminosilanes or polydopamine as coupling layers for the attachment of α-bromoisobutyryl bromide (BiBB) and the direct functionalization of surface hydroxyl groups with trichlorosilane-functionalized ATRP initiators all surfaces studied were shown to facilitate the growth of PDEGMA brushes using the same conditions that were reported previously for polymerization on gold. The brush layers obtained were characterized systematically using wetting, ellipsometry, X-ray photoelectron spectroscopy (XPS) as well as atomic force microscopy (AFM) analyses. Selective cell release and separation of PaTu 8988t and NIH 3T3 cells, which are known to exhibit different behavior after temperature drop-induced brush swelling, was observed for all substrates, albeit for different brush thicknesses, implying variations in initiator and also PDEGMA grafting density. The successful modification of biomedically relevant materials (Ti and TCPS) implies that the previously reported stem cell purification and selective cell release of various cell types, which is facilitated by PDEGMA brushes, can be realized and consequently scaled up in the future. [ABSTRACT FROM AUTHOR]

By analyzing patients treated with adoptive immune cell therapies, various immune cell phenotypes have been found in the starting and infused materials as determinants of sustained remission. The isolation of these specific phenotypes for clinical use requires current Good Manufacturing Practice (cGMP)-compliant cell-sorting technologies with multiparameter selection capabilities. Here, we developed a cGMP-requirement-applicable fully closed cell sorter that has a suction mechanism and multiparameter detection using two laser optical settings. Negative pressure generated by a change in the chamber volume at a sorting point allows the isolation of cells of interest with high viability and purity. Our study demonstrated that this microfluidic sorter enables the isolation of cells of interest at an effective rate of 7,000 sorts per second on average. A purity of 85.5% and 77.1% effective yield with 93.7% viability was obtained when applying a target population of 35.9% in total (lymphocyte+CD8+) at 15,000 events per second (2 × 107 cells/mL). The sorted gene-modified T cells maintain largely unaltered proliferation, antigen recognition, cytokine release, and cytotoxicity functionalities.

Flatworms depend on stem cells for continued tissue growth and renewal during their life cycles, making these cells valuable drug targets. While neoblasts are extensively characterized in the free-living planarian Schmidtea mediterranea, and similar stem cells have been characterized in the trematode Schistosoma mansoni, their identification and characterization in cestodes is just emerging. Since stem cells are generally affected by irradiation, in this work we used this experimental approach to study the stem cells of the model cestode Mesocestoides corti. We found that gamma irradiation produces a dose-dependent decrease in proliferative cells, requiring higher doses than in other flatworms to completely abolish proliferation. The treatment results in the downregulation of candidate marker genes. Transcriptomic studies reveal that several genes downregulated after irradiation are conserved with other flatworms, and are related to cell cycle, DNA replication and repair functions. Furthermore, proliferative cells were isolated by cell sorting and also characterized transcriptomically. We found that the set of genes characteristic of proliferative cells agrees well with those downregulated during irradiation, and have a significant overlap with those expressed in planarian neoblasts or S. mansoni stem cells. Our study highlights that conserved mechanisms of stem cell biology may be functional in flatworms, suggesting that these could be relevant targets to evaluate in the control of parasitic species.

BACKGROUND: We have successfully isolated and cultured neonatal mouse cerebrospinal fluid-contacting neurons in vitro, but there is no study that reports an effective method for isolating and culturing high-purity adult mouse cerebrospinal fluid-contacting neurons. There is no study on whether the self-renewal ability of cerebrospinal fluid-contacting neurons changes with age. OBJECTIVE: To establish a method for isolating and culturing high-purity adult mouse cerebrospinal fluid-contacting neurons in vitro, and to characterize the self-renewal ability of adult mouse cerebrospinal fluid-contacting neurons and neonatal mouse cerebrospinal fluid-contacting neurons in vitro. METHODS: Primary cells containing cerebrospinal fluid-contacting neurons were isolated from the cervical medulla of adult mouse (3 months of age) in adherent culture and transfected with lentivirus fused with multimodal imaging genes. High-purity adult mouse cerebrospinal fluid-contacting neurons were obtained by puromycin screening in suspension culture in complete medium. The expression of neural stem cell markers Nestin and SOX2 was detected by immunofluorescence in adult mouse cerebrospinal fluid-contacting neurons, and the ability of adult mouse cerebrospinal fluid-contacting neurons to form spheres and pass on in vitro was observed. An equal number (5×10³ /mL) of passage 3 adult mouse and neonatal mouse cerebrospinal fluid-contacting neurons were divided into two groups under the same conditions and inoculated into ultra-low adhesion plates containing complete medium in suspension culture at 5% CO2, 37°C thermostat, respectively. The self-renewal capacity of adult mouse and neonatal mouse cerebrospinal fluid-contacting neurons was characterized by in vitro spheroid formation, CCK8 assay, qPCR, and western blot assay. RESULTS AND CONCLUSION: (1) High-purity cerebrospinal fluid-contacting neurons were successfully isolated from adult mouse, which expressed Nestin and SOX2 in vitro, and were able to form neurospheres and pass on continuously. (2) The in vitro self-renewal ability of cerebrospinal fluid-contacting neurons in adult mouse was significantly weaker than that of neonatal mouse, and the neurospheres formed by day 4 of cell culture in neonatal mouse were about 150 μm in diameter, whereas the neurospheres formed by adult mouse tactile neurons were only 40 μm in diameter (P < 0.000 1). (3) CCK8 proliferation assay showed that the proliferative activity of adult mouse cerebrospinal fluid-contacting neurons was significantly weaker than that of neonatal mouse at all time points after culture (P < 0.000 1). (4) qPCR and western blot assay revealed that the mRNA (P < 0.000 1) and protein expression levels (P < 0.01) of Nestin and SOX2 in cerebrospinal fluid-contacting neurons of adult mouse were significantly decreased compared with those of neonatal mouse. (5) The above results indicated that the self-renewal ability of cerebrospinal fluid-contacting neurons in adult mouse was significantly weaker than that of neonatal mouse in vitro. [ABSTRACT FROM AUTHOR]

Cultured meat technologies leverage the proliferation and differentiation of animal-derived stem cells ex vivo to produce edible tissues for human consumption in a sustainable fashion. However, skeletal muscle is a dynamic and highly complex tissue, involving the interplay of numerous mono- and multinucleated cells, including muscle fibers, satellite cells (SCs) and fibro-adipogenic progenitors (FAPs), and recreation of the tissue in vitro thus requires the characterization and manipulation of a broad range of cell types. Here, we use a single-cell RNA sequencing approach to characterize cellular heterogeneity within bovine muscle and muscle-derived cell cultures over time. Using this data, we identify numerous distinct cell types, and develop robust protocols for the easy purification and proliferation of several of these populations. We note overgrowth of undesirable cell types within heterogeneous proliferative cultures as a barrier to efficient cultured meat production, and use transcriptomics to identify conditions that favor the growth of SCs in the context of serum-free medium. Combining RNA velocities computed in silico with time-resolved flow cytometric analysis, we characterize dynamic subpopulations and transitions between active, quiescent, and committed states of SCs, and demonstrate methods for modulation of these states during long-term proliferative cultures. This work provides an important reference for advancing our knowledge of bovine skeletal muscle biology, and its application in the development of cultured meat technologies.

Ongoing, early-stage clinical trials illustrate the translational potential of human pluripotent stem cell (hPSC)-based cell therapies in Parkinson's disease (PD). However, an unresolved challenge is the extensive cell death following transplantation. Here, we performed a pooled CRISPR-Cas9 screen to enhance postmitotic dopamine neuron survival in vivo. We identified p53-mediated apoptotic cell death as a major contributor to dopamine neuron loss and uncovered a causal link of tumor necrosis factor alpha (TNF-α)-nuclear factor κB (NF-κB) signaling in limiting cell survival. As a translationally relevant strategy to purify postmitotic dopamine neurons, we identified cell surface markers that enable purification without the need for genetic reporters. Combining cell sorting and treatment with adalimumab, a clinically approved TNF-α inhibitor, enabled efficient engraftment of postmitotic dopamine neurons with extensive reinnervation and functional recovery in a preclinical PD mouse model. Thus, transient TNF-α inhibition presents a clinically relevant strategy to enhance survival and enable engraftment of postmitotic hPSC-derived dopamine neurons in PD. [Display omitted] • CRISPR-Cas9 screen identifies p53 in restricting in vivo dopamine neuron survival • TNF-α-NF-κB pathway mediates p53-dependent dopamine neuron death • Cell surface marker screen to enrich human dopamine neurons for translational use • Approved TNF-α inhibitor rescues dopamine neuron survival with in vivo function Transient TNF-α inhibition overcomes a p53-dependent program that restricts the survival of grafted dopamine neurons and offers a clinically relevant strategy to enhance survival and promote functional dopamine neuronal engraftment in cell-based therapies for Parkinson's disease. [ABSTRACT FROM AUTHOR]

Graphical Abstract

Here, we developed a straightforward methodology to generate TCRαβ negative (allogeneic) cells for CAR-T cell therapies. With an early and transient expression of an anti-CD3 CAR in the engineered donor T cells, we programmed these cells to self-eliminate the TCR+ cell population and obtained an ultrapure TCRαβ– population (99–99.9%) at the end of the CAR-T production. This novel and easy-to-implement procedure preserves the production yield and cell fitness and has the potential to streamline the manufacturing of “off-the-shelf” CAR T-cell therapies.

Problem: Since not too many human uterus cervical squamous cell carcinoma (CSCC) cell lines in existence, efficient isolation, culture, and purification protocols for primary CSCC cells were optimized as a tool for the study of uterus CSCC. Method of study: The protocols for partial multiple enzymatic digestion and explant cell culture were combined and then the resulting mixed cell component cultures were purified by magnetic‐activated cell sorting. Colony‐forming assay was utilized for detection of cell carcinogenesis potential, and immunofluorescence was used to detect protein expression of CSCC. Finally, flow cytometry (FCM) was performed to analyze cancer stem cells (CSCs) phenotypic markers as well as programmed cell death ligand 1(PD‐L 1). Results: Freshly isolated cells containing tumor cells and cancer‐associated fibroblasts (CAFs) efficiently proliferate to 85% confluence on a 6 cm petri dish in 5‐7 days. Anti‐epithelial cell adhesion molecule antibody (EpCAM) microbeads were used to successfully separate a homogeneous subpopulation of epithelial tumor cells. Both EpCAM+ and EpCAM− cell subpopulations were able to be passaged more than 30 times. Proportions of tumor cell populations expressed CSCs markers such as CD133, CD24, aldehyde dehydrogenase 1 (ALDH1), and CD44. The vimentin+ & EpCAM− population, defined with CAFs, could express CD146 mesenchymal stem cells marker. Meanwhile, PD‐L 1 was identified in most subpopulation of CD44+ cells at low passage numbers. Conclusion: Efficient isolation, culture, and purification protocols for primary CSCC cells were successfully built. Additionally, the profiling of CSCs cell markers might provide promising therapeutic targets and clinic strategies. [ABSTRACT FROM AUTHOR]

Image, graphical abstract Progress in cell purification technology is critical to increase the availability of viable cells for therapeutic, diagnostic, and research applications. A variety of techniques are now available for cell separation, ranging from non-affinity methods such as density gradient centrifugation, dielectrophoresis, and filtration, to affinity methods such as chromatography, two-phase partitioning, and magnetic-/fluorescence-assisted cell sorting. For clinical and analytical procedures that require highly purified cells, the choice of cell purification method is crucial, since every method offers a different balance between yield, purity, and bioactivity of the cell product. For most applications, the requisite purity is only achievable through affinity methods, owing to the high target specificity that they grant. In this review, we discuss past and current methods for developing cell-targeting affinity ligands and their application in cell purification, along with the benefits and challenges associated with different purification formats. We further present new technologies, like stimuli-responsive ligands and parallelized microfluidic devices, towards improving the viability and throughput of cell products for tissue engineering and regenerative medicine. Our comparative analysis provides guidance in the multifarious landscape of cell separation techniques and highlights new technologies that are poised to play a key role in the future of cell purification in clinical settings and the biotech industry. Technologies for cell purification have served science, medicine, and industrial biotechnology and biomanufacturing for decades. This review presents a comprehensive survey of this field by highlighting the scope and relevance of all known methods for cell isolation, old and new alike. The first section covers the main classes of target cells and compares traditional non-affinity and affinity-based purification techniques, focusing on established ligands and chromatographic formats. The second section presents an excursus of affinity-based pseudo-chromatographic and non-chromatographic technologies, especially focusing on magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). Finally, the third section presents an overview of new technologies and emerging trends, highlighting how the progress in chemical, material, and microfluidic sciences has opened new exciting avenues towards high-throughput and high-purity cell isolation processes. This review is designed to guide scientists and engineers in their choice of suitable cell purification techniques for research or bioprocessing needs. [ABSTRACT FROM AUTHOR]

Cell alterations during isolation and preparation for flow cytometry cell sorting by antibodies, temperature, homogenization, buffer composition and mitogens are well known. In contrast, little is known about cell alteration caused by the instrument or the sorting process itself. We systematically evaluated cellular responses to different sorter‐induced physical forces. In summary, flow cytometry cell‐sorting induced forces can affect cellular signaling cascades, especially the MAPK p38. Functional assays, related to the p38 MAPK pathway, of human primary T cells after flow cytometry sorting did lead to minor physiological modulation but no functional impairments. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry. [ABSTRACT FROM AUTHOR]

Introduction: In addition to being frequently used in transplant studies, olfactory ensheathing cells (OECs) within the olfactory mucosa (OM) and olfactory bulb (OB), may be used as carriers to deliver therapeutic agents to specific target areas. These cells that encompass the unmyelinated axons of the olfactory neurons, have an accessible source for isolation, high migratory capacity and ability to secrete anti-inflammatory and neurotrophic factors. The mentioned features making them reasonable candidates for cell therapy. Although recent advancements in OEC isolation techniques heralded a new era in the field, the viability rate and efficacy of purification are still need to be considered. The most widely used OEC isolation strategies can be classified according to their adhesive properties, especially for isolating OECs from the OB. Considering the invasive nature of harvesting OECs from the human OB, highly efficient purification from the OM may be clinically beneficial. Methods and Materials: In this study, we isolated and compared OECs from both OB and OM of rat, due to their different adherence characteristics. By immunocytochemistry and western blot analysis, specific markers of OEC cells were identified with NGFRp75 and S100β antibodies. OECs morphology and viability were tracked over time using microscopy and MTT assay. Results: We discovered that using our proposed strategy, OECs can be purified from the OM as efficiently as the OB. OECs from both sources exhibited high levels of NGFRp75 and S100β expression, although the S100β expression was higher in OEC preparations derived from the OM (P<0.05). Furthermore, there was no significant difference in cell viability between the two sources. Conclusion: Therefore, OECs extracted from the OM are obtained in a non-invasive manner and are clinically suitable for transplantation studies. [ABSTRACT FROM AUTHOR]

The authors report on a mild, label-free, and fast method for the separation of human umbilical vein endothelial cells (HUVEC), which are relevant cells, whose use is not limited to studies of endothelial dysfunction, from cocultures with macrophages to afford HUVEC in ≈100% purity. Poly(di(ethylene glycol)methyl ether methacrylate) (PDEGMA) brushes with a dry thickness of (5 ± 1) nm afford the highly effective one-step separation by selective HUVEC detachment, which is based on the brushes' thermoresponsive behavior. Below the thermal transition at 32 °C the brushes swells and desorbs attached proteins, resulting in markedly decreased cell adhesion. Specifically, HUVEC and macrophages, which are differentiated from THP-1 monocytes, are seeded and attached to PDEGMA brushes at 37°C. After decreasing the temperature to 22°C, HUVEC shows a decrease in their cell area, while the macrophages are not markedly affected by the temperature change. After mild flushing with a cell culture medium, the HUVEC can be released from the surface and reseeded again with ≈100% purity on a new surface. With this selective cell separation and removal method, it is possible to separate and thereby purify HUVEC from macrophages without the use of any releasing reagent or expensive labels, such as antibodies., (© 2023 The Authors. Macromolecular Bioscience published by Wiley-VCH GmbH.)

Transcriptomic analyses of germ cells at different stages of differentiation have shed light on the transcriptional and post-transcriptional mechanisms regulating gene expression that ensure the correct progression of spermatogenesis and male fertility. In this chapter, we describe a method to isolate meiotic and post-meiotic germ cells, based on gravimetric sedimentation, starting from a testicular germ cell suspension isolated from a single adult mouse. We also describe how to assess the purity and quality of the collected fractions of germ cells and how to optimize the extraction from these samples of RNA for subsequent RNA-sequencing experiment. In our experience, this protocol is suitable for germ cell isolation and transcriptomic analysis for mouse models with spermatogenic defects, overcoming the limits that reduced fertility poses to the obtaining of experimental animals., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Fast and accurate interrogation of complex samples containing diseased cells or pathogens is important to make informed decisions on clinical and public health issues. Inertial microfluidics has been increasingly employed for such investigations to isolate target bioparticles from liquid samples with size and/or deformability-based manipulation. This phenomenon is especially useful for the clinic, owing to its rapid, label-free nature of target enrichment that enables further downstream assays. Inertial microfluidics leverages the principle of inertial focusing, which relies on the balance of inertial and viscous forces on particles to align them into size-dependent laminar streamlines. Several distinct microfluidic channel geometries (e.g., straight, curved, spiral, contraction-expansion array) have been optimized to achieve inertial focusing for a variety of purposes, including particle purification and enrichment, solution exchange, and particle alignment for on-chip assays. In this review, we will discuss how inertial microfluidics technology has contributed to improving accuracy of various assays to provide clinically relevant information. This comprehensive review expands upon studies examining both endogenous and exogenous targets from real-world samples, highlights notable hybrid devices with dual functions, and comments on the evolving outlook of the field.

Medullary thymic epithelial cells (mTEC) are bona fide antigen-presenting cells that play a crucial role in the induction of T-cell tolerance. By their unique ability to express a broad range of tissue-restricted self-antigens, mTEC control the clonal deletion (also known as negative selection) of potentially hazardous autoreactive T cells and the generation of Foxp3 + regulatory T cells. Here, we describe a protocol to assess major histocompatibility complex (MHC) class II antigen-presentation capacity of mTEC to CD4 + T cells. We detail the different steps of thymus enzymatic digestion, immunostaining, cell sorting of mTEC and CD4 + T cells, peptide-loading of mTEC, and the co-culture between these two cell types. Finally, we describe the flow cytometry protocol and the subsequent analysis to assess the activation of CD4 + T cells. This rapid co-culture assay enables the evaluation of the ability of mTEC to present antigens to CD4 + T cells in an antigen-specific context. Key features • This protocol builds upon the method used by Lopes et al. (2018 and 2022) and Charaix et al. (2022). • This protocol requires transgenic mice, such as OTIIx Rag2 -/- mice and the cognate peptide OVA 323-339 , to assess mTEC antigen presentation to CD4 + T cells. • This requires specific equipment such as a Miltenyi Biotec AutoMACS ® Pro Separator, a BD FACSAria TM III cell sorter, and a BD ® LSR II flow cytometer., Competing Interests: Competing interestsAuthors declare no competing interests., (©Copyright : © 2023 The Authors; This is an open access article under the CC BY license.)

Background Satellite glial cells (SGCs) envelope the neuronal somas in the dorsal root ganglia (DRG) and are believed to provide important neuronal support. Animal models of peripheral nerve injury, diabetes or chemotherapy all demonstrate activation of SGCs, suggesting important physiological roles for SGCs in various states of peripheral neuropathy. However, the biology of these glial cells is only poorly characterized under normal as well as pathological conditions due to suboptimal isolation methods. New method The method presented here allows complete dissociation and isolation of highly pure SGCs from rat DRGs by fluorescence-activated cell sorting (FACS) using SGC-specific antibodies. The method further allows purification of high-quality RNA from the fixed and permeabilized cells. Results The purified RNA shows very little degradation, demonstrated by RNA integrity number (RIN) analysis with an average value of 8. The purified RNA, therefore, lends itself very well to downstream applications such as qPCR and transcriptome analysis. Comparison with existing methods Primary SGC cultures have previously been established for in vitro studies. Unfortunately, SGCs quickly change morphology and gene expression in vitro , complicating biologically meaningful interpretation of the obtained results. In contrast, this method allows the investigation of SGC gene regulation in vivo by isolation of high-quality RNA. Conclusions This method enables investigation of SGC transcriptional response in vivo by isolation and analysis of mRNA expression, allowing a more detailed investigation of SGC biology under normal as well as pathological conditions. [ABSTRACT FROM AUTHOR]

Cultured meat technologies leverage the proliferation and differentiation of animal-derived stem cells ex vivo to produce edible tissues for human consumption in a sustainable fashion. However, skeletal muscle is a dynamic and highly complex tissue, involving the interplay of numerous mono- and multinucleated cells, including muscle fibers, satellite cells (SCs) and fibro-adipogenic progenitors (FAPs), and recreation of the tissue in vitro thus requires the characterization and manipulation of a broad range of cell types. Here, we use a single-cell RNA sequencing approach to characterize cellular heterogeneity within bovine muscle and muscle-derived cell cultures over time. Using this data, we identify numerous distinct cell types, and develop robust protocols for the easy purification and proliferation of several of these populations. We note overgrowth of undesirable cell types within heterogeneous proliferative cultures as a barrier to efficient cultured meat production, and use transcriptomics to identify conditions that favor the growth of SCs in the context of serum-free medium. Combining RNA velocities computed in silico with time-resolved flow cytometric analysis, we characterize dynamic subpopulations and transitions between active, quiescent, and committed states of SCs, and demonstrate methods for modulation of these states during long-term proliferative cultures. This work provides an important reference for advancing our knowledge of bovine skeletal muscle biology, and its application in the development of cultured meat technologies., Competing Interests: TM, RD, LS, LM, RH, MG, and JF are employees of Mosa Meat B.V. MP is co-founder and stakeholder of Mosa Meat B.V. Study was funded by Mosa Meat B.V. Mosa Meat B.V. has patents on serum-free proliferation medium (WO2021158103), and serum-free myogenic (WO2022114955) and adipogenic differentiation media (WO2023003470). The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Messmer, Dohmen, Schaeken, Melzener, Hueber, Godec, Didoss, Post and Flack.)

By analyzing patients treated with adoptive immune cell therapies, various immune cell phenotypes have been found in the starting and infused materials as determinants of sustained remission. The isolation of these specific phenotypes for clinical use requires current Good Manufacturing Practice (cGMP)-compliant cell-sorting technologies with multiparameter selection capabilities. Here, we developed a cGMP-requirement-applicable fully closed cell sorter that has a suction mechanism and multiparameter detection using two laser optical settings. Negative pressure generated by a change in the chamber volume at a sorting point allows the isolation of cells of interest with high viability and purity. Our study demonstrated that this microfluidic sorter enables the isolation of cells of interest at an effective rate of 7,000 sorts per second on average. A purity of 85.5% and 77.1% effective yield with 93.7% viability was obtained when applying a target population of 35.9% in total (lymphocyte+CD8+) at 15,000 events per second (2 × 10 7 cells/mL). The sorted gene-modified T cells maintain largely unaltered proliferation, antigen recognition, cytokine release, and cytotoxicity functionalities., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

The use of human mesenchymal stem cells (hMSC) in clinical applications has been increasing over the last decade. However, to be applied in a clinical setting hMSC need to comply with specific requirements in terms of identity, potency and purity. This study reports the improvement of established tangential flow filtration (TFF)-based washing strategies, further increasing hMSC purity, using negative mode expanded bed adsorption (EBA) chromatography with a new multimodal prototype matrix based on core–shell bead technology. The matrix was characterized and a stable, expanded bed could be obtained using standard equipment adapted from what is used for conventional packed bed chromatography processes. The effect of different expansion rates on cell recovery yield and protein removal capacity was assessed. The best trade-off between cell recovery (89%) and protein clearance (67%) was achieved using an intermediate expansion bed rate (1.4). Furthermore, we also showed that EBA chromatography can be efficiently integrated on the already established process for the downstream processing (DSP) of hMSC, where it improved the washing efficiency more than 10-fold, recovering approximately 70% of cells after global processing. This strategy showed not to impact cell viability (>95%), neither hMSC's characteristics in terms of morphology, immunophenotype, proliferation, adhesion capacity and multipotent differentiation potential. [ABSTRACT FROM AUTHOR]

Purification of defined cell populations from mixed primary cell sources is essential for many biomedical and biotechnological applications but often very difficult to accomplish due to missing specific surface markers. In this study, we developed a new approach for efficient cell population separation based on the specific membrane fusion characteristics of distinct cell types upon treatment with fusogenic liposomes. When such liposomes are conjugated with biotin, specific cell populations can be efficiently surface functionalized by biotin after liposomal treatment while other populations remain unlabeled. Due to the high affinity of biotin for avidin-like proteins, biotin functionalized cells are ideal targets for conjugation of e.g. avidin tagged magnetic beads, fluorophores or antibodies with bioanalytical relevance. Here, based on the differential biotinylation of distinct cell populations high quality separation of cardiac fibroblasts from myocytes, and cerebromicrovascular endothelial cells from fibroblasts was successfully established. [ABSTRACT FROM AUTHOR]

Monocytes are key innate effector cells and their phenotype and function may be a useful biomarker of disease state or therapeutic response. However, for such an assay to be clinically feasible it needs to be simple and reproducible, which this study aimed to address. Peripheral blood mononuclear cells (PBMC) 2 isolated from whole blood using Histopaque-1077 or cell preparation tubes (CPT) showed no difference in the ex vivo monocyte activation marker expression or in vitro responses; however, a delayed isolation using CPT significantly altered ex vivo and in vitro phenotypes and responses . Furthermore, purification of monocytes using CD14 + microbeads resulted in a loss of CD14 low CD16 + monocytes compared to PBMC samples. Thus, the use of CPT reduced complexity and time compared to Histopaque, and PBMC isolation allowed the analysis of all 3 major monocyte subsets. Finally, because the delayed isolation of PBMC from CPT significantly altered monocytes, time delays should be standardized. [ABSTRACT FROM AUTHOR]

The study of CNS glial cell function requires experimental methods to detect, purify, and manipulate each cell population with fidelity and specificity. With the identification and cloning of cell- and stage-specific markers, glial cell analysis techniques have grown beyond physical methods of tissue dissociation and cell culture, and become highly specific with immunoselection of cell cultures in vitro and genetic targeting in vivo . The unique plasticity of glial cells offers the potential for cell replacement therapies in neurological disease that utilize neural cells derived from transplanted neural stem and progenitor cells. In this mini-review, we outline general physical and genetic approaches for macroglial cell generation. We summarize cell culture methods to obtain astrocytes and oligodendrocytes and their precursors, from developing and adult tissue, as well as approaches to obtain human neural progenitor cells through the establishment of stem cells. We discuss popular targeting rodent strains designed for cell-specific detection, selection and manipulation of neuroglial cell progenitors and their committed progeny. Based on shared markers between astrocytes and stem cells, we discuss genetically modified mouse strains with overlapping expression, and highlight SOX-expressing strains available for targeting of stem and progenitor cell populations. We also include recently established mouse strains for detection, and tag-assisted RNA and miRNA analysis. This discussion aims to provide a brief overview of the rapidly expanding collection of experimental approaches and genetic resources for the isolation and targeting of macroglial cells, their sources, progeny and gene products to facilitate our understanding of their properties and potential application in pathology. [ABSTRACT FROM AUTHOR]

Background aims. Despite promising advances in cellular therapies, it will be difficult to fully test or implement new therapies until advances are made in the processes for cell preparation. This study describes the use of an advanced prototype of a flow-cytometry cell purification system constructed for operation in a clinical environment to prepare regulatory T cells defined as CD4+/CD25brightCD127neg/low Methods. The sort performance of the Gigasort system was directly compared with available droplet sorters using mixtures of highly fluorescent and non-fluorescent 5-|tm polystyrene particles. CD4 -enriched cell preparations were processed with the use of a sterile, disposable fluid handling unit with a chip containing parallel micro-fluidic-based sorters. Results. Similar purity and sort efficiency as found with droplet sorters were obtained with the 24-channel chip sorter system. Starting with 450 million fresh peripheral blood mononuclear cells, 150,000 to 1.7 million cells that were, on average, 85% FoxP3-positive and 97% viable, were obtained in <4 h. Conclusions. This study presents a technology adapted to regulatory requirements for clinical cell purification and that achieves high throughput and cell-friendly conditions by use of a microfluidic chip with 24 parallel microsorters, providing a rapid, sterile method of purifying regulatory T cells accurately and with excellent viability. [ABSTRACT FROM AUTHOR]

In medical diagnostics, detection of cells exhibiting specific phenotypes constitutes a paramount challenge. Detection technology must ensure efficient isolation of (often rare) targets while eliminating nontarget background cells. Technologies exist for such investigations, but many require high levels of expertise, expense, and multistep protocols. Increasing automation, miniaturization, and availability of such technologies is an aim of microfluidic lab-on-a-chip strategies. To this end, we present an integrated, dual-force cellular separation strategy using centrifugo-magnetophoresis. Whole blood spiked with target cells is incubated with (super-)paramagnetic microparticles that specifically bind phenotypic markers on target cells. Under rotation, all cells sediment into a chamber located opposite a co-rotating magnet. Unbound cells follow the radial vector, but under the additional attraction of the lateral magnetic field, bead-bound target cells are deflected to a designated reservoir. This multiforce separation is continuous and low loss. We demonstrate separation efficiently up to 92% for cells expressing the HIV/AIDS relevant epitope (CD4) from whole blood. Such highly selective separation systems may be deployed for accurate diagnostic cell isolations from biological samples such as blood. Furthermore, this high efficiency is delivered in a cheap and simple device, thus making it an attractive option for future deployment in resource-limited settings. [ABSTRACT FROM PUBLISHER]

Human pluripotent stem cells (hPSCs) are a promising source of cells for clinical applications, such as transplantation of clinically engineered tissues and organs, and drug discovery programs due to their ability to self-renew and to be differentiated into cells from the three embryonic germ layers. In this study, the differentiation of two hPSC-lines into neural precursors (NPs) was accomplished with more than 80 % efficiency, by means of the dual-SMAD inhibition protocol, based on the use of two small molecules (SB431542 and LDN193189) to generate Pax6 and Nestin-positive neural entities. One of the major hurdles related to the in vitro generation of PSC-derived populations is the tumorigenic potential of cells that remain undifferentiated. These remaining hPSCs have the potential to generate teratomas after being transplanted, and may interfere with the outcome of in vitro differentiation protocols. One strategy to tackle this problem is to deplete these 'contaminating' cells during the differentiation process. Magnetic activated cell sorting (MACS) was used for the first time for purification of hPSC-derived NPs after the neural commitment stage using anti-Tra-1-60 micro beads for negative selection of the unwanted hPSCs. The depletion had an average efficiency of 80.4 ± 5 % and less than 1.5 % of Tra-1-60 positive cells were present in the purified populations. After re-plating, the purified neural precursors maintained their phenotype, and the success of the preparative purification with MACS was further confirmed with a decrease of 94.3 % in the number of Oct4-positive proliferating hPSC colonies. Thus, the integration of the MACS depletion step with the neural commitment protocol paves the way towards the establishment of a novel bioprocess for production of purified populations of hPSC-derived neural cells for different applications. [ABSTRACT FROM AUTHOR]

As a powerful tool of cell screening and cell purification, we developed a novel method to kill adherent cells as cultured on a substrate by micro-projection of incoherent visible light. To kill the cells by the mild light irradiated by electrically controllable micro-projection systems currently available, we introduced the assist of the photo-responsive culture substrates functionalized with a photo-acid-generating polymer. In clear contrast to the existing laser-based methods requiring point scanning, areal micro-prjection of blue light with the wavelength 436 nm killed many CHO-K1 cells at a time in the irradiated area on the substrate. The effect of the photo-generated acid was so confined that selective killing of targeted cells was achieved without critical damage to the neighboring cells. Further, we demonstrated the photo-selective killing of the adherent cells after preliminarily patterning through the photo-induced removal of cell adhesion-inhibiting polymer. Biotechnol. Bioeng. 2013; 110: 348-352. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

To circumvent the difficulty of isolating specific cell populations by MACS from dissociated complex animal tissue, when their proportions reached levels similar to that of the background, we developed the "Three-step MACS" strategy. Cells of interest are defined by their expression of a particular gene(s) of interest rather by than their natural cell surface markers or size. A two-component transgenic cell surface protein, for two sequential rounds of MACS, is expressed under the promoter control of the endogenous gene of interest by means of gene targeting and the generation of transgenic tissue. An initial step to remove dead cells is also used. Here, we describe proof-of-concept experiments, using the biotin acceptor peptide (BAP)-low-affinity nerve growth factor receptor as the two-component protein. The first component, the BAP, can be biotinylated in specific subsets of cells expressing a particular gene by expressing the biotinylating enzyme, hBirA = humanized BirA (hBirA), under the promoter control of another gene defining the specific subpopulation. We showed that a rare population of cells (1.1% of the 13.5 days postcoital mouse embryo) could be enriched to a sufficiently high purity (84.4%). From another sample with 0.1% of our cells of interest, we achieved a 40.3% pure sample. The low cost, speed, and technical ease of the Three-step MACS also make it scalable and hence, an ideal method for preparing sufficient quantities of biological samples for sensitive, high-throughput assays. [ABSTRACT FROM AUTHOR]

Abstract: Recent genome-level innovations have enabled the identification of the entire cadre of genes that are expressed in specific tissues at particular developmental times. However, to be informative as to how individual cell types develop, this process relies upon the successful and efficient purification of cells for a particular tissue. Here, we describe a method to isolate cardiac cells from Drosophila embryos. We generated transgenic embryos in which a cardiac-specific enhancer of the Sulphonylurea receptor (Sur) gene drove expression of the green fluorescent protein (GFP) gene. Homogenized embryos were subjected to fluorescence activated cell sorting (FACS), resulting in approximately 50,000 cardiac cells purified. The prevalence of cardiac cells in the purified population was high, based upon a significant enrichment for cardiac-specific marker genes, including Sur and Toll. This enrichment also enabled the identification of cardiac-specific alternatively spliced isoforms of the Zasp66 gene. In the future, this approach can be used to describe the cardiac transcriptome of Drosophila at distinct stages of embryonic development. [Copyright &y& Elsevier]

Sponges (Porifera), as the best known source of bioactive marine natural products in metazoans, play a significant role in marine drug discovery and development. As sessile filter-feeding animals, a considerable portion of the sponge biomass can be made of endosymbiotic and associated microorganisms. Understanding the cellular origin of targeted bioactive compounds from sponges is therefore important not only for providing chemotaxonomic information but also for defining the bioactive production strategy in terms of sponge aquaculture, cell culture, or fermentation of associated bacteria. The two alkaloids debromohymenialdisine (DBH) and hymenialdisine (HD), which are cyclin-dependent kinase inhibitors with pharmacological activities for treating osteoarthritis and Alzheimer's disease, have been isolated from the sponge Axinella sp. In this study, the cellular localization of these two alkaloids was determined through the quantification of these alkaloids in different cell fractions by high-performance liquid chromatography (HPLC). First, using a differential centrifugation method, the dissociated cells were separated into different groups according to their sizes. The two bioactive alkaloids were mainly found in sponge cells obtained from low-speed centrifugation. Further cell purifications were accomplished by a newly developed multi-step protocol. Four enriched cell fractions (C1, C2, C3, and C4) were obtained and subjected to light and transmission electron microscopy, cytochemical staining, and HPLC quantification. Compared to the low concentrations in other cell fractions, DBH and HD accounted for 10.9% and 6.1%, respectively, of dry weight in the C1 fraction. Using the morphological characteristics and cytochemical staining results, cells in the C1 fraction were speculated to be spherulous cells. This result shows that DBH and HD in Axinella sp. are located in sponge cells and mostly stored in spherulous cells. [ABSTRACT FROM AUTHOR]

Due to their pluripotency and their self-renewal capacity, human pluripotent stem cells (hPSC) provide fascinating perspectives for biomedical applications. In the long term, hPSC-derived tissue-specific cells will constitute an important source for cell replacement therapies in non-regenerative organs. These therapeutic approaches, however, will critically depend on the purity of the in vitro differentiated cell populations. In particular, remaining undifferentiated hPSC in a transplant can induce teratoma formation. In order to address this challenge, we have developed a laser-based method for the ablation of hPSC from differentiating cell cultures. Specific antibodies were directed against the hPSC surface markers tumor related antigen (Tra)-1-60 and Tra-1-81. These antibodies, in turn, were targeted with nanogold particles. Subsequent laser exposure resulted in a 98,9 ± 0,9% elimination of hPSCs within undifferentiated cell cultures. In order to study potential side effects of laser ablation on cells negative for Tra-1-60 and Tra-1-81, hPSC were mixed with GFP-positive hPSC-derived neural precursors (hESCNP) prior to ablation. These studies showed efficient elimination of hPSC while co-treated hESCNP maintained their normal proliferation and differentiation potential. In vivo transplantation of treated and untreated mixed hPSC/hESCNP cultures revealed that laser ablation can dramatically reduce the risk of teratoma formation. Laser-assisted photothermolysis thus represents a novel contact-free method for the efficient elimination of hPSC from in vitro differentiated hPSC-derived somatic cell populations. [ABSTRACT FROM AUTHOR]

Abstract: Background: Eosinophils are key effector cells in allergy and in other inflammatory diseases. Although they carry out their function in the tissues, no efficient method exists allowing for consistent purification of tissue eosinophils for culture. Rather, studies rely mainly on peripheral blood eosinophils. This study aimed to determine the most efficient protocol for purifying eosinophils from nasal polyp tissue. Methods: Nasal polyps were obtained from patients undergoing surgical polypectomy. The polyps were minced and enzymatically digested. Surface receptor analysis was performed by flow cytometry. In order to obtain optimal purification, the nasal polyp cell suspension was subjected to two methods of purification: 1) positive magnetic selection of CCR3+cells, or 2) negative selection using CD3/CD14/CD16 magnetic beads. Enriched tissue eosinophils were cultured with or without IL-3, IL-5 or GM-CSF, and their survival was evaluated by flow cytometry. Results: Tissue-derived eosinophils exhibited surface expression of NEC2, DNAM-1, NTBa, 2B4, and CD300a comparable to similarly prepared eosinophils obtained from the peripheral blood of the same patients. Positive selection consistently yielded eosinophils of high purity (>90%) with 63% viability. In contrast, negative selection yielded better viability (88%), reduced purity (66%), and could be utilized for in vitro activation experiments. Conclusion: Eosinophils can be purified from nasal polyps. Negative selection appears to be advantageous due to improved viability of the eosinophils, which may be cultured and activated in vitro. This methodology is an important advance in studying tissue eosinophils for further investigations on inflammatory tissue responses. [Copyright &y& Elsevier]

Objective: To analyze the constitutive production of epithelial neutrophil activating peptide 78 (ENA-78) and interleukin-8 (IL-8) by epithelial cells and the response of these cells to cytokine stimulation.Design: In vitro study using eutopic endometrial tissue.Setting: University hospital.Patient(s): Cycling women undergoing laparoscopy for reasons of infertility or unexplained abdominal pain.Intervention(s): Isolation of epithelial and stromal cells from endometrium, immunocytochemical characterization and separate culture of these cells in presence of IL-1, tumor necrosis factor alpha (TNF-alpha), and interferon-gamma.Main Outcome Measure(s): Quantitation of IL-8 and ENA-78 released into the medium by ELISA. Polymerase chain reaction was used to demonstrate the presence of ENA-78 in the cell lysate.Result(s): High purity of the endometrial epithelial cell preparation before culture was demonstrated by the lack of immunocytochemical staining for CD10. Stromal cell preparations were CD10 positive and cytokeratin negative. Stromal cells produced ENA-78 and IL-8 under cytokine stimulation, and epithelial cells were found not only to produce these markers in the absence of cytokine stimulation, but also to increase this output in the presence of IL-1beta or of TNF-alpha plus interferon-gamma.Conclusion(s): This response may be an important angiogenic step in the early stages in the pathogenesis of endometriosis. [ABSTRACT FROM AUTHOR]

Objective: Establishment of an efficient method of preparing human kidney single cell suspension, using a very small amount of tissue puncture. Methods: Samples of human kidney tissue puncture were cut into pieces, and then 80 μL of the digestive enzyme were added to each punctured tissue to induce enzymatic digestion. The enzyme combination is composed of collagenases, DNase and hyaluronidase and the sample was incubated 20 min at 37°C. The obtained cell suspension was filtered through a 70 μm cell strainer, centrifuged at 300 g for 5 min and the supernatant was removed, then the pellet was resuspended in 3 ml of DMEM (Dulbecco's Modified Eagle's Medium). Cell suspension was sorted and purified by flow sorting to remove dead cells and obtain a cell suspension with higher viability rate. Results: We found that 1) diverse single cells of human kidney can be obtained by the digestive enzyme, as observed under the light microscope, with different sizes, normal cell morphology and good dispersion. 2) (2-3) × 10 6 single cells can be extracted from one fresh punctured kidney tissue of about 10 mg, with a cell viability rate of more than 80%. Conclusion: In this work we generated a comprehensive and high-resolution single-cell method, which is simple and efficient for preparing single cell suspension from a minimal amount of human kidney tissue. This method can facilitate the study of renal cell biology and the pathogenesis of kidney diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yaigoub, Tirichen, Xin, Shi, Wu, Li and Li.)

In humans, studies of the erythroid cell lineage are hampered by difficulties in obtaining sufficient numbers of erythroid progenitors. In fact, these progenitors in bone marrow or peripheral blood are scarce and no specific antibodies are available. We describe a new method which allows proliferation in liquid culture of large numbers of pure normal human erythroid progenitors. CD34+ cells were cultured for 7 d in serum-free conditions with the cytokine mixture interleukin (IL)-3/IL-6/stem cell factor (SCF). This resulted in cell expansion and the appearance of a high proportion of CD36+ cells which were purified on day 7. Methylcellulose clones from these cells were composed of 96.6% late BFU-E and 3.4% CFU-GM. These CD36+ cells could be recultured with the same cytokine mixture plus or minus erythropoietin (Epo) for a further 2–7 d. In both conditions further amplification of CD36+ cells was observed, but Epo induced a more dramatic cell expansion. Glycophorin-positive mature cells appeared only in the presence of Epo, and terminal red cell differentiation was observed after 7 d of secondary culture. Cells obtained from adult CD34+ progenitors mostly contained adult haemoglobin, whereas cord blood-derived cells contained equal proportions of adult and fetal haemoglobin. Activation of STAT5 and tyrosine phosphorylation of the Epo receptor and JAK2 were observed after Epo stimulation of these cells. This new method represents a straightforward alternative to the procedures previously described for the purification of normal erythroid progenitors and is useful in the study of erythropoietic regulation. [ABSTRACT FROM AUTHOR]

Fast and accurate interrogation of complex samples containing diseased cells or pathogens is important to make informed decisions on clinical and public health issues. Inertial microfluidics has been increasingly employed for such investigations to isolate target bioparticles from liquid samples with size and/or deformability-based manipulation. This phenomenon is especially useful for the clinic, owing to its rapid, label-free nature of target enrichment that enables further downstream assays. Inertial microfluidics leverages the principle of inertial focusing, which relies on the balance of inertial and viscous forces on particles to align them into size-dependent laminar streamlines. Several distinct microfluidic channel geometries (e.g., straight, curved, spiral, contraction-expansion array) have been optimized to achieve inertial focusing for a variety of purposes, including particle purification and enrichment, solution exchange, and particle alignment for on-chip assays. In this review, we will discuss how inertial microfluidics technology has contributed to improving accuracy of various assays to provide clinically relevant information. This comprehensive review expands upon studies examining both endogenous and exogenous targets from real-world samples, highlights notable hybrid devices with dual functions, and comments on the evolving outlook of the field. [ABSTRACT FROM AUTHOR]

Flow cytometry and fluorescence-activated cell sorting have become invaluable tools to analyze and isolate specific cell populations in a wide range of biomedical research and clinical applications. In countless approaches worldwide, scientists are using single cell analyses to better understand the significance and variation within different cellular populations, and fluorescence-activated cell sorting has become a major technique for cell isolation in both basic and clinical research. However, majority of available cell sorters are pressurized, droplet-based systems, which apply significant environmental pressure and shear stress to cells during sorting. Recently, the flow cytometry community has become increasingly aware about the potential negative effects this could have on sorted cells and the term "sorter induced cell stress" (SICS) has been proposed. However, up to date only a limited number of studies have investigated the effects of cell sorting on cell viability and function. Therefore, solid data on the effects of sheath pressure and nozzle size on survival and function of sorted cells are surprisingly rare. With this in mind, we sorted "CD4+" T-cells and "live" cells from human peripheral blood mononuclear cells (PBMCs) at different sort conditions and analyzed their quality before and after sorting in a series of assays. Here we present our findings in reference to cell viability and cell proliferation following sorting on different instruments (BD FACSAria III SORP and BD FACSJazz), utilizing different nozzle sizes (70 to 100 μm) and sheath pressure settings (20 to 70 psi). The results show no significant differences in cell viability and proliferation after the different tested sort conditions, but rather differences between individual experiments. These findings are evaluated and their potential significance in cell sorting experiments is discussed. • Analyzing the effect of sorter induced stress on cell viability and proliferation • Human PBMCs were sorted using different sorters and nozzles • Viability and proliferation of sorted cells were studied by 7AAD and CFSE • Sorting at different conditions did not reduce cell viability • Cell proliferation was not altered after different sort conditions for up to 7 days [ABSTRACT FROM AUTHOR]

Although antibodies are routinely used to label and isolate a desired cell type from a more complex mixture of cells, via either fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS), such antibody labeling is not easily reversible. We describe an FACS and MACS compatible method to reversibly label and purify cells using aptamers. Magnetic beads loaded with the epidermal growth factor receptor (EGFR)-binding antagonistic aptamer E07 specifically isolated EGFR-expressing cells, and pure, label-free cells were recovered via treatment with an "antidote" oligonucleotide complementary to the aptamer. Additionally, while FACS sorting cells with E07 or EGFR antibody yielded EGFR(+) cells with impeded EGFR signaling, stripping off the aptamer via antidote treatment restored receptor function, returning cells to their native state, which was not possible with the antibody. The ability to reversibly label or isolate cells without compromising their function is a valuable, versatile tool with important implications for both the laboratory and clinic. • Aptamers can be used to reversibly label and purify cells via FACS or MACS • EGFR aptamer-magnetic bead MACS isolates EGFR+ cells out of complex cell mixtures • An oligonucleotide "antidote" strips off aptamers to recover label-free cells • EGFR aptamer FACS sorting and antidote removal restores native receptor function Cell purification methods rely upon poorly reversible antibody labeling of desired cells or upon depletion of unwanted cells. Powell Gray et al. demonstrate that aptamers can be used to positively purify cells and, unlike antibodies, yield cells in their native state using antidote-mediated reversal of aptamer cell-surface binding. [ABSTRACT FROM AUTHOR]

The isolation of a highly pure and healthy population of macrophages from the CNS in a short time is essential for studying the role of these cells in injury and disease. Current methods rely either on the use of gradients and/or sorting, processes that result either in impure, reduced viability and/or altered populations of cells. Furthermore, RNA extraction after immunopanning is often difficult. Here, a technique combining both gradient isolation and immunopanning to generate highly pure cultures of primary macrophages isolated from the injured adult CNS in <2 h is described. An optimized protocol of a commercially-available RNA extraction kit is also outlined for the isolation of highly pure RNA. A hybridoma cell-line producing CD11b antibody was used to recover activated (CD11b+) macrophages/microglia from the CNS after injury in <2 h with >95% purity. • Immunopanning with a hybridoma-derived CD11b antibody is used to isolate macrophages and activated microglia from the CNS • Isolation requires no new equipment, is cost-efficient, results in a highly pure cell population, and takes <2 h to complete • Commercially-available RNA extraction kits have been optimized to obtain RNA suitable for molecular biology analysis [ABSTRACT FROM AUTHOR]

Here, we developed a straightforward methodology to generate TCRαβ negative (allogeneic) cells for CAR-T cell therapies. With an early and transient expression of an anti-CD3 CAR in the engineered donor T cells, we programmed these cells to self-eliminate the TCR+ cell population and obtained an ultrapure TCRαβ - population (99-99.9%) at the end of the CAR-T production. This novel and easy-to-implement procedure preserves the production yield and cell fitness and has the potential to streamline the manufacturing of "off-the-shelf" CAR T-cell therapies., (Copyright © 2020 Juillerat, Tkach, Yang, Boyne, Valton, Poirot and Duchateau.)

A treatment for intractable diseases is expected to be the replacement of damaged tissues with products from human induced pluripotent stem cells (hiPSCs). Target cell purification is a critical step for realizing hiPSC-based therapy. Here, we found that hiPSC-derived ocular cell types exhibited unique adhesion specificities and growth characteristics on distinct E8 fragments of laminin isoforms (LNE8s): hiPSC-derived corneal epithelial cells (iCECs) and other non-CECs rapidly adhered preferentially to LN332/411/511E8 and LN211E8, respectively, through differential expression of laminin-binding integrins. Furthermore, LN332E8 promoted epithelial cell proliferation but not that of the other eye-related cells, leading to non-CEC elimination by cell competition. Combining these features with magnetic sorting, highly pure iCEC sheets were fabricated. Thus, we established a simple method for isolating iCECs from various hiPSC-derived cells without using fluorescence-activated cell sorting. This study will facilitate efficient manufacture of iCEC sheets for corneal disease treatment and provide insights into target cell-specific scaffold selection., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Hepatocytes derived from human pluripotent stem cells (hPSCs) are promising candidates for cell therapy and drug discovery. However, it remains challenging to efficiently purify hepatocytes from undesired cell types after differentiation and to accurately monitor grafted cells after transplantation. Indocyanine Green (ICG), an FDA-approved, near-infrared (NIR) dye, has been used for various clinical purposes and is exclusively taken up by hepatocytes. However, ICG has a long emission wavelength (λ em > 800 nm) that is beyond the detection range of fluorescence-activated cell sorting (FACS) systems. Moreover, it is easily eliminated from hepatocytes, hindering its application for NIR imaging. Here, we designed and synthesized two different probes based on the properties of ICG; 1) hepatocyte purifying agent (HPA, λ em = 562 nm) for in vitro sorting and 2) hepatocyte imaging agent (HIA, λ em = 817 nm) for efficient in vivo NIR imaging. We obtained highly enriched populations of hPSC-derived hepatocytes (hPSC-Heps) from various hPSC lines using HPA probe-based FACS purification. In addition, HIA labelling and NIR imaging allowed the direct visualization and tracking of grafted hPSC-Heps in animals with liver injuries. These results demonstrated that these two probes could be used as powerful tools with hPSC-Heps in both cell replacement therapy and drug screening. [ABSTRACT FROM AUTHOR]