Understanding of mouse and human bladder at single-cell resolution: integrated analysis of trajectory and cell-cell interactive networks based on multiple scRNA-seq datasets.

OBJECTIVES: To elaborately decipher the mouse and human bladders at single-cell levels. MATERIALS AND METHODS: We collected more than 50,000 cells from multiple datasets and created, up to date, the largest integrated bladder datasets. Pseudotime trajectory of urothelium and interstitial cells, as well as dynamic cell-cell interactions, was investigated. Biological activity scores and different roles of signaling pathways between certain cell clusters were also identified. RESULTS: The glucose score was significantly high in most urothelial cells, while the score of H3 acetylation was roughly equally distributed across all cell types. Several genes via a pseudotime pattern in mouse (Car3, Dkk2, Tnc, etc.) and human (FBLN1, S100A10, etc.) were discovered. S100A6, TMSB4X, and typical uroplakin genes seemed as shared pseudotime genes for urothelial cells in both human and mouse datasets. In combinational mouse (n = 16,688) and human (n = 22,080) bladders, we verified 1,330 and 1,449 interactive ligand-receptor pairs, respectively. The distinct incoming and outgoing signaling was significantly associated with specific cell types. Collagen was the strongest signal from fibroblasts to urothelial basal cells in mouse, while laminin pathway for urothelial basal cells to smooth muscle cells (SMCs) in human. Fibronectin 1 pathway was intensely sent by myofibroblasts, received by urothelial cells, and almost exclusively mediated by SMCs in mouse bladder. Interestingly, the cell cluster of SMCs 2 was the dominant sender and mediator for Notch signaling in the human bladder, while SMCs 1 was not. The expression of integrin superfamily (the most common communicative pairs) was depicted, and their co-expression patterns were located in certain cell types (eg, Itgb1 and Itgb4 in mouse and human basal cells). CONCLUSIONS: This study provides a complete interpretation of the normal bladder at single-cell levels, offering an in-depth resource and foundation for future research.

Genome profiles of pathologist-defined cell clusters by multiregional LCM and G&T-seq in one triple-negative breast cancer patient.

Pathological examination is the gold standard for cancer diagnosis, and breast tumor cells are often found in clusters. We report a case study on one triple-negative breast cancer (TNBC) patient, analyzing tumor development, metastasis, and prognosis with simultaneous DNA and RNA sequencing of pathologist-defined cell clusters from multiregional frozen sections. The cell clusters are isolated by laser capture microdissection (LCM) from primary tumor tissue, lymphatic vessels, and axillary lymph nodes. Data are reported for a total of 97 cell clusters. A combination of tumor cell-cluster clonality and phylogeny reveals 3 evolutionarily distinct pathways for this patient, each associated with a unique mRNA signature, and each correlated with disparate survival outcomes. Hub gene analysis indicates that extensive downregulation of ribosomal protein mRNA is a potential marker of poor prognosis in breast cancer.

Differentiation of Human-Induced Pluripotent Stem Cell-Derived Endocrine Progenitors to Islet-like Cells Using a Dialysis Suspension Culture System.

The production of functional islet-like cells from human-induced pluripotent stem cells (hiPSCs) is a promising strategy for the therapeutic use and disease modeling for type 1 diabetes. However, the production cost of islet-like cells is extremely high due to the use of expensive growth factors for differentiation. In a conventional culture method, growth factors and beneficial autocrine factors remaining in the culture medium are removed along with toxic metabolites during the medium change, and it limits the efficient utilization of those factors. In this study, we demonstrated that the dialysis suspension culture system is possible to reduce the usage of growth factors to one-third in the differentiation of hiPSC-derived endocrine progenitor cells to islet-like cells by reducing the medium change frequency with the refinement of the culture medium. Furthermore, the expression levels of hormone-secretion-related genes and the efficiency of differentiation were improved with the dialysis suspension culture system, possibly due to the retaining of autocrine factors. In addition, we confirmed several improvements required for the further study of the dialysis culture system. These findings showed the promising possibility of the dialysis suspension culture system for the low-cost production of islet-like cells.

Programmable Aggregation of Artificial Cells with DNA Signals.

Cell aggregation is a complex behavior that is closely related to the viability, differentiation, and migration of cells. An effort to create synthetic analogs could lead to considerable advances in cell physiology and biophysics. Rendering and modulating such a dynamic artificial cell system require mechanisms for receiving, transducing, and transmitting intercellular signals, yet effective tools are limited at present. Here we construct synthetic cells from engineered lipids and show their programmable aggregation behaviors using DNA oligonucleotides as signaling molecules. The artificial cells have transmembrane channels made of DNA origami that are used to recognize and process intercellular signals. We demonstrate that multiple small vesicles aggregate onto a giant vesicle after a transduction of external DNA signals by an intracellular enzyme and that the small vesicles dissociate when receiving "release" signals. This work provides new possibilities for building synthetic protocells capable of chemical communication and coordination.

Single-cell RNA landscape of intratumoral heterogeneity and immunosuppressive microenvironment in advanced osteosarcoma.

Osteosarcoma is the most frequent primary bone tumor with poor prognosis. Through RNA-sequencing of 100,987 individual cells from 7 primary, 2 recurrent, and 2 lung metastatic osteosarcoma lesions, 11 major cell clusters are identified based on unbiased clustering of gene expression profiles and canonical markers. The transcriptomic properties, regulators and dynamics of osteosarcoma malignant cells together with their tumor microenvironment particularly stromal and immune cells are characterized. The transdifferentiation of malignant osteoblastic cells from malignant chondroblastic cells is revealed by analyses of inferred copy-number variation and trajectory. A proinflammatory FABP4+ macrophages infiltration is noticed in lung metastatic osteosarcoma lesions. Lower osteoclasts infiltration is observed in chondroblastic, recurrent and lung metastatic osteosarcoma lesions compared to primary osteoblastic osteosarcoma lesions. Importantly, TIGIT blockade enhances the cytotoxicity effects of the primary CD3+ T cells with high proportion of TIGIT+ cells against osteosarcoma. These results present a single-cell atlas, explore intratumor heterogeneity, and provide potential therapeutic targets for osteosarcoma.

TICAM2-related pathway mediates neutrophil exhaustion.

Pathogenic inflammation and immune suppression are the cardinal features that underlie the pathogenesis of severe systemic inflammatory syndrome and sepsis. Neutrophil exhaustion may play a key role during the establishment of pathogenic inflammation and immune suppression through elevated expression of inflammatory adhesion molecules such as ICAM1 and CD11b as well as immune-suppressors such as PD-L1. However, the mechanism of neutrophil exhaustion is not well understood. We demonstrated that murine primary neutrophils cultured in vitro with the prolonged lipopolysaccharides (LPS) stimulation can effectively develop an exhaustive phenotype resembling human septic neutrophils with elevated expression of ICAM1, CD11b, PD-L1 as well as enhanced swarming and aggregation. Mechanistically, we observed that TICAM2 is involved in the generation of neutrophil exhaustion, as TICAM2 deficient neutrophils have the decreased expression of ICAM1, CD11b, PD-L1, and the reduced aggregation following the prolonged LPS challenge as compared to wild type (WT) neutrophils. LPS drives neutrophil exhaustion through TICAM2 mediated activation of Src family kinases (SFK) and STAT1, as the application of SFK inhibitor Dasatinib blocks neutrophil exhaustion triggered by the prolonged LPS challenge. Functionally, TICAM2 deficient mice were protected from developing severe systemic inflammation and multi-organ injury following the chemical-induced mucosal damage. Together, our data defined a key role of TICAM2 in facilitating neutrophil exhaustion and that targeting TICAM2 may be a potential approach to treating the severe systemic inflammation.

Neutrophil Swarming in Damaged Tissue Is Orchestrated by Connexins and Cooperative Calcium Alarm Signals.

Neutrophils are major inflammatory cells that rapidly infiltrate wounds to provide antimicrobial functions. Within the damaged tissue, neutrophil migration behavior often switches from exploratory patrolling to coordinated swarming, giving rise to dense clusters that further disrupt tissue architecture. This aggregation response is self-organized by neutrophil paracrine chemoattractant signaling (most notably of the inflammatory mediator leukotriene B4 [LTB4]). The coordination mechanism and possible evolutionary benefits of neutrophil swarms are elusive. Here, we show that neutrophil swarms require mutual reinforcement of damage signaling at the wound core. New biosensors and live imaging in zebrafish revealed that neutrophil chemoattractant synthesis is triggered by a sustained calcium flux upon contact with necrotic tissue that requires sensing of the damage signal ATP. This "calcium alarm" signal rapidly propagates in the nascent neutrophil cluster in a contact-dependent manner via connexin-43 (Cx43) hemichannels, which are mediators of active ATP release. This enhances chemoattractant biosynthesis in the growing cluster, which is instrumental for coordinated motion and swarming. Inhibition of neutrophil Cx43 compromises clearance of wound-colonizing P. aeruginosa bacteria and exacerbates infection-induced morbidity. Thus, cooperative production of alarm signals among pioneer clustering neutrophils fuels the growth of dense antimicrobial cell masses that effectively seal off breached tissue barriers from opportunistic pathogens.

Characterization of three-dimensional rat central nervous system culture maturation, with applications to monitor cholinergic integrity.

Studying age-related neuropathologies in vitro requires a three-dimensional (3D) culture system presenting mature phenotypes. In this study, we aimed to determine whether aged reaggregate cultures physiologically represent mature brain tissue. Results support that embryo-derived rat central nervous system (CNS) reaggregate cultures develop into mature-like tissues, comparable to in vivo maturation, including the following characteristics: (a) progressive reduction in cell proliferation (reduced anti-Ki-67 immunoreactivity), (b) progressive restriction of long neurite growth potential (as explant cultures), and (c) increased and sustained synaptic enzyme (acetylcholine esterase, AChE) activity. The acquisition of mature-like reaggregate cultures has allowed us to pursue the hypothesis that the physiological integrity of 3D CNS cultures may be monitored by synaptic enzyme activity. To assess this hypothesis, mature-like reaggregates were exposed to H2 O2 , glutamate, or amyloid ß(1-42); each resulted in diminished AChE activity. H2 O2 exposure resulted in nuclear fragmentation. Glutamate and amyloid ß(1-42) exposure resulted in acetylcholine content reduction. Simultaneous reduction of AChE activity and acetylcholine content verified diminished cholinergic integrity. This scheme exploiting synapse enzyme activity of mature-like 3D CNS tissue is therefore applicable to age-related neuropathology research including in vitro screening of conditions potentially affecting synapse integrity, including the promotion of dementia.

Endothelial intercellular cell adhesion molecule 1 contributes to cell aggregate formation in CHO cells cultured in serum-free media.

Chinese hamster ovary (CHO) cells have been adapted to grow in serum-free media and in suspension culture to facilitate manufacturing needs. Some CHO cell lines, however, tend to form cell aggregates while being cultured in suspension. This can result in reduced viability and capacity for single cell cloning (SCC) via limiting dilution, and process steps to mitigate cell aggregate formation, for example, addition of anti-cell-aggregation agents. In this study, we have identified endothelial intercellular cell adhesion molecule 1 (ICAM-1) as a key protein promoting cell aggregate formation in a production competent CHO cell line, which is prone to cell aggregate formation. Knocking out (KO) the ICAM-1 gene significantly decreased cell aggregate formation in the culture media without anti-cell-aggregation reagent. This trait can simplify the process of transfection, selection, automated clone isolation, and so on. Evaluation in standard cell line development of ICAM-1 KO and wild-type CHO hosts did not reveal any noticeable impacts on titer or product quality. Furthermore, analysis of a derived nonaggregating cell line showed significant reductions in expression of cell adhesion proteins. Overall, our data suggest that deletion of ICAM-1 and perhaps other cell adhesion proteins can reduce cell aggregate formation and improve clonality assurance during SCC.

Tumor cell­fibroblast heterotypic aggregates in malignant ascites of patients with ovarian cancer.

Ascitic multicellular aggregates (MCAs) promote peritoneal metastasis of ovarian cancer. The aim of the present study was to elucidate the role of cancer­associated fibroblasts (CAFs) in MCA formation and metastasis in patients with high­grade serous ovarian cancer (HGSOC). Immunohistochemistry was used to identify the cell phenotypes and the presence of CAFs in ascitic MCAs. The role of CAFs in tumor­cell MCA formation was assessed by co­culture in suspension. Primary ascitic tumor cells and omental CAFs were used to generate ex vivo MCAs in hanging drops, and the invasiveness of MCAs was evaluated by mesothelial clearance and adhesion assays in vitro and in vivo. MCAs containing CAFs and tumor cells were identified in the ascitic fluid. CAFs facilitated tumor cell aggregation and compaction to form MCAs, and enhanced the mesothelial clearance and adhesion abilities of tumor­cell MCAs. These findings suggest that ascitic CAFs promote peritoneal metastasis by forming heterotypic aggregates with tumor cells, and that they may serve as potential targets for the treatment of HGSOC.

Relation between cell-bound exopolysaccharide production via plasmid-encoded genes and rugose colony morphology in the probiotic Lactobacillus brevis KB290.

The probiotic Lactobacillus brevis KB290 is a natural producer of cell-bound exopolysaccharide (EPS), and the plasmid-encoded glycosyltransferase genes are responsible for this EPS production. KB290 forms unique rugose colonies inside an agar medium; this characteristic is useful for detecting and enumerating KB290 in the gut or feces. However, the genetic elements associated with this morphology remain unclear. Here, we aimed to investigate the relation between the plasmid eps genes and rugose colony morphology in KB290. The plasmid-cured mutants formed smooth colonies, and the rugose colony morphology was restored after complementation with the eps genes. The eps genes were successfully cloned and expressed in other L. brevis and L. plantarum strains. In these transformant strains, the presence of the EPS, consisting of glucose and N-acetylglucosamine, correlated with rugose colonies, indicating that EPS is responsible for rugose colony formation. To the best of our knowledge, this is the first report identifying the genetic factors influencing rugose colonies in Lactobacillus strains. This rugose colony formation may serve as a useful selective marker for KB290 in routine laboratory and research settings and can be used to detect the spontaneous loss of plasmids in this strain.

Prognostic relevance of the Golgi mannosidase MAN1A1 in ovarian cancer: impact of N-glycosylation on tumour cell aggregation.

BACKGROUND: Maturation of complex N-glycans involves the action of Golgi mannosidases and plays a major role in cancer progression. We recently showed a favourable prognostic role of α-mannosidase MAN1A1 in breast cancer mainly caused by alteration of certain adhesion molecules. METHODS: We analysed the protein expression of MAN1A1 in ovarian cancer (n = 204) using western blot and studied the impact of MAN1A1 itself and of MAN1A1-related glycosylation on the prognostic relevance of two adhesion molecules. Functional consequences of mannosidase inhibition using kifunensine and MAN1A1 knock out were investigated in ovarian cancer cells in vitro. RESULTS: Patients with high MAN1A1 expression in tumours showed significantly shorter RFS than those with low-MAN1A1 levels. Moreover, high MAN1A1 expression correlated significantly with advanced stage, lymph node involvement and distant metastasis. Further, the glycosylated adhesion molecule ALCAM reveals a significant adverse prognostic effect only in the presence of high MAN1A1 expression. In spheroid-formation assays, mannosidase inhibition and especially MAN1A1 knock out led to strong reduction of tumour cell aggregation. CONCLUSIONS: Our study demonstrates the unfavourable prognostic role of MAN1A1 in ovarian cancer, probably caused by an altered ability of spheroid formation, and the strong influence of this glycosylation enzyme on the prognostic impact of ALCAM.

GLI1 activation by non-classical pathway integrin αvß3/ERK1/2 maintains stem cell-like phenotype of multicellular aggregates in gastric cancer peritoneal metastasis.

Peritoneal metastasis is one of the most important causes of postoperative death in patients with gastric cancer, and the exact mechanism remains unclear. The proliferation of multicellular aggregates of exfoliated malignant gastric cells in the abdominal cavity is the focus of current research. However, the mechanism how gastric cancer multicellular aggregates survive remains unclear. In this study, we demonstrated that multicellular aggregates of exfoliated gastric cancer cells in the abdominal cavity expressed a stem cell-Like phenotype. We found that Integrin αvß3 not only mediated adhesion of gastric cancer multicellular aggregates to form independent functional units, but also maintained their stem cell-like phenotype by the non-classical pathway Integrin αvß3/ERK1/2/GLI1. In addition, ERK1/2 directly regulates the transcriptional activity of GLI1. GLI1 is a key effector of the Integrin αvß3 pathway in regulating stem cell-like phenotype in multicellular aggregates. Our data indicates that although there is a crosstalk between the non-classical Integrin αvß3 pathway and the classical Hedgehog pathway, the activation of GLI1 is almost independent of the Hedgehog pathway in multicellular aggregates of gastric cancer cells. Our study provides a basis for blocking GLI1 activity in the prevention and treatment of peritoneal metastases of gastric cancer.

Transcriptomic analysis of 3D Cardiac Differentiation of Human Induced Pluripotent Stem Cells Reveals Faster Cardiomyocyte Maturation Compared to 2D Culture.

Human induced pluripotent stem cells (hiPSCs) represent an almost limitless source of cells for disease modelling and drug screening applications. Here we established an efficient and robust 3D platform for cardiomyocyte (CMs) production from hiPSCs, solely through small-molecule-based temporal modulation of the Wnt signalling, which generates more than 90% cTNT+ cells. The impact of performing the differentiation process in 3D conditions as compared to a 2D culture system, was characterized by transcriptomic analysis by using data collected from sequential stages of 2D and 3D culture. We highlight that performing an initial period of hiPSC aggregation before cardiac differentiation primed hiPSCs towards an earlier mesendoderm lineage differentiation, via TGF-ß/Nodal signaling stabilization. Importantly, it was also found that CMs in the 3D microenvironment mature earlier and show an improved communication system, which we suggested to be responsible for a higher structural and functional maturation of 3D cardiac aggregates.

A telomerase with novel non-canonical roles: TERT controls cellular aggregation and tissue size in Dictyostelium.

Telomerase, particularly its main subunit, the reverse transcriptase, TERT, prevents DNA erosion during eukaryotic chromosomal replication, but also has poorly understood non-canonical functions. Here, in the model social amoeba Dictyostelium discoideum, we show that the protein encoded by tert has telomerase-like motifs, and regulates, non-canonically, important developmental processes. Expression levels of wild-type (WT) tert were biphasic, peaking at 8 and 12 h post-starvation, aligning with developmental events, such as the initiation of streaming (~7 h) and mound formation (~10 h). In tert KO mutants, however, aggregation was delayed until 16 h. Large, irregular streams formed, then broke up, forming small mounds. The mound-size defect was not induced when a KO mutant of countin (a master size-regulating gene) was treated with TERT inhibitors, but anti-countin antibodies did rescue size in the tert KO. Although, conditioned medium (CM) from countin mutants failed to rescue size in the tert KO, tert KO CM rescued the countin KO phenotype. These and additional observations indicate that TERT acts upstream of smlA/countin: (i) the observed expression levels of smlA and countin, being respectively lower and higher (than WT) in the tert KO; (ii) the levels of known size-regulation intermediates, glucose (low) and adenosine (high), in the tert mutant, and the size defect's rescue by supplemented glucose or the adenosine-antagonist, caffeine; (iii) the induction of the size defect in the WT by tert KO CM and TERT inhibitors. The tert KO's other defects (delayed aggregation, irregular streaming) were associated with changes to cAMP-regulated processes (e.g. chemotaxis, cAMP pulsing) and their regulatory factors (e.g. cAMP; acaA, carA expression). Overexpression of WT tert in the tert KO rescued these defects (and size), and restored a single cAMP signaling centre. Our results indicate that TERT acts in novel, non-canonical and upstream ways, regulating key developmental events in Dictyostelium.

Staphylococcus aureus aggregation in the plasma fraction of silkworm hemolymph.

Staphylococcus aureus formed bacterial aggregates in the plasma fraction of the hemolymph of silkworm, the larva of Bombyx mori, in a growth-dependent manner. The addition of arabinose or galactose inhibited the formation of S. aureus aggregates in the silkworm plasma. Formation of the bacterial aggregates depended on S. aureus genes required for the synthesis of bacterial surface polysaccharides-ypfP and ltaA, which are involved in lipoteichoic acid synthesis, and the tagO gene, which is involved in wall teichoic acid synthesis. These findings suggest that S. aureus forms bacterial aggregates in the silkworm plasma via bacterial surface teichoic acids.

Transcriptional Programming of Normal and Inflamed Human Epidermis at Single-Cell Resolution.

Perturbations in the transcriptional programs specifying epidermal differentiation cause diverse skin pathologies ranging from impaired barrier function to inflammatory skin disease. However, the global scope and organization of this complex cellular program remain undefined. Here we report single-cell RNA sequencing profiles of 92,889 human epidermal cells from 9 normal and 3 inflamed skin samples. Transcriptomics-derived keratinocyte subpopulations reflect classic epidermal strata but also sharply compartmentalize epithelial functions such as cell-cell communication, inflammation, and WNT pathway modulation. In keratinocytes, ∼12% of assessed transcript expression varies in coordinate patterns, revealing undescribed gene expression programs governing epidermal homeostasis. We also identify molecular fingerprints of inflammatory skin states, including S100 activation in the interfollicular epidermis of normal scalp, enrichment of a CD1C+CD301A+ myeloid dendritic cell population in psoriatic epidermis, and IL1ßhiCCL3hiCD14+ monocyte-derived macrophages enriched in foreskin. This compendium of RNA profiles provides a critical step toward elucidating epidermal diseases of development, differentiation, and inflammation.

Non-local Parabolic and Hyperbolic Models for Cell Polarisation in Heterogeneous Cancer Cell Populations.

Tumours consist of heterogeneous populations of cells. The sub-populations can have different features, including cell motility, proliferation and metastatic potential. The interactions between clonal sub-populations are complex, from stable coexistence to dominant behaviours. The cell-cell interactions, i.e. attraction, repulsion and alignment, processes critical in cancer invasion and metastasis, can be influenced by the mutation of cancer cells. In this study, we develop a mathematical model describing cancer cell invasion and movement for two polarised cancer cell populations with different levels of mutation. We consider a system of non-local hyperbolic equations that incorporate cell-cell interactions in the speed and the turning behaviour of cancer cells, and take a formal parabolic limit to transform this model into a non-local parabolic model. We then investigate the possibility of aggregations to form, and perform numerical simulations for both hyperbolic and parabolic models, comparing the patterns obtained for these models.

Aggregation of human dental pulp cells into 3D spheroids enhances their migration ability after reseeding.

Multicellular three-dimensional (3D) spheroids allow intimate cell-cell communication and cell-extracellular matrix interaction. Thus, 3D cell spheroids better mimic microenvironment in vivo than two-dimensional (2D) monolayer cultures. The purpose of this study was to evaluate the behaviors of human dental pulp cells (DPCs) cultured on chitosan and polyvinyl alcohol (PVA) membranes. The protein expression of hypoxia-inducible factor 1-α (HIF-1α) and vascular endothelial growth factor (VEGF), and the migration ability of the DPCs from 2D versus 3D environments were investigated. The results showed that both chitosan and PVA membranes support DPCs aggregation to form multicellular spheroids. In comparison to 2D cultures on tissue culture polystyrene, DPC spheroids exhibited higher protein expression of HIF-1α and VEGF. The treatment with YC-1 (inhibitor to HIF-1α) blocked the upregulation of VEGF, indicating a downstream event to HIF-1α expression. When DPC spheroids were collected and subjected to the transwell assay, the cells growing outward from 3D spheroids showed greater migration ability than those from 2D cultures. Moreover, DPCs aggregation and spheroid formation on chitosan membrane were abolished by Y-27632 (inhibitor to Rho-associated kinases), whereas the inhibitory effect did not exist on PVA membrane. This suggests that the mechanism regulating DPCs aggregation and spheroid formation on chitosan membrane is involved with the Rho-associated kinase signaling pathway. In summary, the multicellular spheroid structure was beneficial to the protein expression of HIF-1α and VEGF in DPCs and enhanced the migration ability of the cells climbing from spheroids. This study showed a new perspective in exploring novel strategies for DPC-based research and application.

Novel fluid shear-based dissociation device for improved single cell dissociation of spheroids and cell aggregates.

Biological industries commonly rely on bioreactor systems for the large-scale production of cells. Cell aggregation, clumping, and spheroid morphology of certain suspension cells make their large-scale culture challenging. Growing stem cells as spheroids is indispensable to retain their stemness, but large spheroids (>500 µm diameter) suffer from poor oxygen and nutrient diffusion, ultimately resulting in premature cell death in the centers of the spheroids. Despite this, most large-scale bioprocesses do not have an efficient method for dissociating cells into single cells, but rely on costly enzymatic dissociation techniques. Therefore, we tested a proof-of-concept fluid shear-based mechanical dissociator that was designed to dissociate stem cell spheroids and aggregates. Our prototype was able to dissociate cells while retaining high viability and low levels of apoptosis. The dissociator also did not impact long-term cell growth or spheroid formation. Thus, the dissociator introduced here has the potential to replace traditional dissociation methods. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:293-298, 2018.