Adherent Cell Population Research Articles

Abstract 3200: Assessing the utility of cell membrane lipid-extracted nanoliposomes (CLENs) as a targeted drug delivery system for pancreatic cancer

Abstract Background: Pancreatic cancer is a challenging type of cancer to treat as evidenced by a 9% 5-year survival and a dismal rate of mortality. Nanoparticles represent a revolutionary treatment method, primarily due to characteristics including cellular absorption, boosting therapeutic effectiveness, and mitigating side effects through localized actions. For nanomedicine, optimizing liposome formulations is crucial for achieving the highest level of specificity in drug targeting. This study aims to evaluate cellular interaction of pancreatic cancer lipid-extract modified nanoliposomes when compared to unmodified varieties in vitro. Method: Cell membrane lipid-extracted nanoliposomes (CLENs) were prepared via the thin film evaporation technique. The composition included various compositions and ratios of DOPC, DOTAP, Cholesterol, DPPE-PEG-5000, and lipid extracts derived from MS1-VEGF target cells. MS1-VEGF was the cellular model of pancreatic cancer used for this study. Cellular binding studies were performed using cellular suspension and adherent cell populations. Studies were performed in 48-well plates under different experimental conditions including alterations in time of incubation, and periods of cellular exposure to dual humidified and oscillation environments in the presence of lipid-extracted (LE) nanoliposomal preparations and conventional controls. Results: The particle size for nanoliposomes employed for this study was approximately 115 ± 0.7 nm for control and 128 ± 2.7 nm for experimental (lipid extract-modified) nanoliposomes. Following cellular exposure to different lipid extract concentrations (typically 0 to 100 nmoles per ml of growth medium), the fluorescence values demonstrated increased cellular uptake with greater LE content for floating and adhered MS1-VEGF cell populations. With the floating cells, LE-modified nanoliposomes demonstrated significantly higher fluorescence intensity values compared to conventional nanoliposomes. Similarly, with MS1-VEGF adherent cells, LE-modified nanoliposomes targeted cells to a greater extent compared to unmodified controls. Conclusions: The increased cellular uptake of CLENs designed for targeting pancreatic tumor cells (both floating and adherent cells) demonstrated an improvement over the use of conventional nanoliposomes under similar experimental conditions. Further research is needed to fully explore the potential use of CLENs for targeting and treatment of pancreatic cancer. Citation Format: Khaznah Alshammari, Sergio Verdery, Sujin George, Robert B Campbell. Assessing the utility of cell membrane lipid-extracted nanoliposomes (CLENs) as a targeted drug delivery system for pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3200.

Deetect: A Deep Learning-Based Image Analysis Tool for Quantification of Adherent Cell Populations on Oxygenator Membranes after Extracorporeal Membrane Oxygenation Therapy.

The strong interaction of blood with the foreign surface of membrane oxygenators during ECMO therapy leads to adhesion of immune cells on the oxygenator membranes, which can be visualized in the form of image sequences using confocal laser scanning microscopy. The segmentation and quantification of these image sequences is a demanding task, but it is essential to understanding the significance of adhering cells during extracorporeal circulation. The aim of this work was to develop and test a deep learning-supported image processing tool (Deetect), suitable for the analysis of confocal image sequences of cell deposits on oxygenator membranes at certain predilection sites. Deetect was tested using confocal image sequences of stained (DAPI) blood cells that adhered to specific predilection sites (junctional warps and hollow fibers) of a phosphorylcholine-coated polymethylpentene membrane oxygenator after patient support (>24 h). Deetect comprises various functions to overcome difficulties that occur during quantification (segmentation, elimination of artifacts). To evaluate Deetects performance, images were counted and segmented manually as a reference and compared with the analysis by a traditional segmentation approach in Fiji and the newly developed tool. Deetect outperformed conventional segmentation in clustered areas. In sections where cell boundaries were difficult to distinguish visually, previously defined post-processing steps of Deetect were applied, resulting in a more objective approach for the resolution of these areas.

Micro-fragmented adipose tissue cellular composition varies by processing device and analytical method

Autologous adipose-derived biologics are of clinical interest based on accessibility of adipose tissue, a rich source of progenitor and immunomodulatory cells. Micro-fragmented adipose tissue (MFAT) preserves the cellular niche within intact extracellular matrix, potentially offering benefit over enzymatically-liberated stromal vascular fraction (SVF), however lack of standardized analyses complicate direct comparison of these products. In this study, MFAT from LipoGems® and AutoPose™ Restore systems, which utilize different washing and resizing methods, was analyzed for cellular content using different techniques. Flow cytometry was performed on SVF, with or without culture, and on the adherent cell population that naturally migrated from undigested MFAT. Cytokine release during culture was also assessed. SVF contained more diverse progenitor populations, while MFAT outgrowth contained lower cell concentrations of predominantly mesenchymal stromal cells (MSC). MSCs were significantly higher in MFAT from the AutoPose System for all analyses, with increased cytokine secretion characterized by high levels of anti-inflammatory and low to non-detectable inflammatory cytokines. These results demonstrate that cellularity depends on MFAT processing methods, and different techniques can be employed to evaluate graft cellularity. Comparisons of cell concentrations determined via these methods could be used to better interpret inter-study variability.

Interaction between Mesenchymal Stem Cells and the Immune System in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease that causes damage to joints. This review focuses on the possibility of influencing the disease through immunomodulation by mesenchymal stem cells (MSCs). There is an occurrence of rheumatoid factor and RA-specific autoantibodies to citrullinated proteins in most patients. Citrulline proteins have been identified in the joints of RA patients, and are considered to be the most suitable candidates for the stimulation of anti-citrulline protein antibodies production. Fibroblast-like proliferating active synoviocytes actively promote inflammation and destruction in the RA joint, in association with pro-inflammatory cells. The inflammatory process may be suppressed by MSCs, which are a population of adherent cells with the following characteristic phenotype: CD105+, CD73+, CD90+, CD45−, CD34− and HLA DR−. Following the stimulation process, MSCs are capable of immunomodulatory action through the release of bioactive molecules, as well as direct contact with the cells of the immune system. Furthermore, MSCs show the ability to suppress natural killer cell activation and dendritic cells maturation, inhibit T cell proliferation and function, and induce T regulatory cell formation. MSCs produce factors that suppress inflammatory processes, such as PGE2, TGF-β, HLA-G5, IDO, and IL-10. These properties suggest that MSCs may affect and suppress the excessive inflammation that occurs in RA. The effect of MSCs on rheumatoid arthritis has been proven to be a suitable alternative treatment thanks to successful experiments and clinical studies.

Abstract 3894: WNT5B in osteosarcoma stem cells

Abstract Osteosarcoma is a rare but deadly pediatric bone cancer. As it stands, there is no targeted therapy available for osteosarcoma. Therefore, patients are left with chemotherapy and surgical resection as their only treatment options. Further, osteosarcoma frequently metastasizes to the lungs and patients with metastatic disease have a dismal overall survival rate of 20% due to the lack of targeted therapy. Therefore, the goal of this study is to understand the role of the WNT5B signaling pathway in osteosarcoma, as it could be a potential therapeutic target. Using RNA sequencing from publicly available data sets and immunohistochemistry on tumor microarrays, we show that WNT5B is overexpressed in a subset of osteosarcoma patients. In these high expressing patients, WNT5B’s overexpression correlates to metastasis and worse overall survival. Due to the significant increase in WNT5B expression between primary tumors and metastasis, we began looking at the stem cell population of osteosarcoma cells. By selecting for the osteosarcoma stem cells using spheroid assays, we show that both protein and mRNA levels of WNT5B are enhanced in the stem cell population compared to the standard adherent cell population. Then, we looked at stemness markers and we show that WNT5B regulates the stemness gene SOX2. We then performed limiting dilution sphere assays to assess the role of WNT5B in sphere forming. We reveal that WNT5B is directly responsible for the sphere forming efficiency, as evidenced by a 50% reduction in spheres formed between 143B control and 143B WNT5B knockdown cells. Additionally, WNT5B drives proliferation and migration of osteosarcoma cancer stem cells as there is significantly decreased sphere size and migratory distance in WNT5B knockdown cells compared to control cells, and there is significantly increased sphere size and migration with the addition of recombinant WNT5B. Through revealing a novel role for WNT5B in osteosarcoma cancer stem cells, we present the WNT5B pathway as a candidate for therapeutically targeting the clinical implications of cancer stem cells, that is, the development of chemoresistance, metastasis and relapse. Citation Format: Rachel S. Perkins, Sarocha Suthon, Gustavo A. Miranda-Carboni, Susan A. Krum. WNT5B in osteosarcoma stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3894.

Buddleoside inhibits TLR4-related pathway in a mouse model of acute liver failure, promotes autophagy, and inhibits inflammation

Purpose: To study the inhibitory influence of buddleoside on TLR4-associated pathway, autophagy and inflammation in a mouse model of acute liver failure (ALF).Methods: Sixty male C57BL/6 mice were assigned to 5 groups: control, model, and three dose-groups of buddleoside, with 12 mice per group. Levels of interleukin (IL)-1, IL-6, TLR4 pathway-associated proteins, and autophagy-related proteins in each group were determined; cell adhesion in each group was also analyzed.Results: Levels of TLR4, MAPK and NF-кB-related pathways in model mice were significantly upregulated, relative to control mice, but they were more down-regulated in the 3 anthocyanin groups than in model group (p < 0.05). There were significantly higher levels of TNF-α, IL- and IL-6 in model mice than in the control group, but they were down-regulated in high-, medium- and low-dose mice, relative to model mice. The population of adherent cells was significantly higher in ALF mice than in controls, butthere were markedly lower numbers of these cells in the 3 anthocyanin-treated mice than in model mice (p < 0.05).Conclusion: Buddleoside mitigates ALF in mice by down-regulating inflammatory factors, reducing serum levels of ALT and AST, and up-regulating autophagy-related protein expressions by activating TLR4/MAPK/NF-кB signaling pathway. Thus, buddleoside may be useful in the treatment of acute liver failure, but this has to be curtained through clinical trials.

Real-time luminescence enables continuous drug–response analysis in adherent and suspension cell lines

ABSTRACT The drug-induced proliferation (DIP) rate is a metric of in vitro drug response that avoids inherent biases in commonly used metrics such as 72 h viability. However, DIP rate measurements rely on direct cell counting over time, a laborious task that is subject to numerous challenges, including the need to fluorescently label cells and automatically segment nuclei. Moreover, it is incredibly difficult to directly count cells and accurately measure DIP rates for cell populations in suspension. As an alternative, we use real-time luminescence measurements derived from the cellular activity of NAD(P)H oxidoreductase to efficiently estimate drug response in both adherent and suspension cell populations to a panel of known anticancer agents. For the adherent cell lines, we collect both luminescence reads and direct cell counts over time simultaneously to assess their congruency. Our results demonstrate that the proposed approach significantly speeds up data collection, avoids the need for cellular labels and image segmentation, and opens the door to significant advances in high-throughput screening of anticancer drugs.

Fibronectin Adherent Cell Populations Derived From Avascular and Vascular Regions of the Meniscus Have Enhanced Clonogenicity and Differentiation Potential Under Physioxia.

The meniscus is composed of an avascular inner region and vascular outer region. The vascular region has been shown to contain a progenitor population with multilineage differentiation capacity. Strategies facilitating the isolation and propagation of these progenitors can be used to develop cell-based meniscal therapies. Differential adhesion to fibronectin has been used to isolate progenitor populations from cartilage, while low oxygen or physioxia (2% oxygen) enhances the meniscal phenotype. This study aimed to isolate progenitor populations from the avascular and vascular meniscus using differential fibronectin adherence and examine their clonogenicity and differentiation potential under hyperoxia (20% oxygen) and physioxia (2% oxygen). Human vascular and avascular meniscus cells were seeded onto fibronectin-coated dishes for a short period and monitored for colony formation under either hyperoxia or physioxia. Non-fibronectin adherent meniscus cells were also expanded under both oxygen tension. Individual fibronectin adherent colonies were isolated and further expanded, until approximately ten population doublings (passage 3), whereby they underwent chondrogenic, osteogenic, and adipogenic differentiation. Physioxia enhances clonogenicity of vascular and avascular meniscus cells on plastic or fibronectin-coated plates. Combined differential fibronectin adhesion and physioxia isolated a progenitor population from both meniscus regions with trilineage differentiation potential compared to equivalent hyperoxia progenitors. Physioxia isolated progenitors had a significantly enhanced meniscus matrix content without the presence of collagen X. These results demonstrate that combined physioxia and fibronectin adherence can isolate and propagate a meniscus progenitor population that can potentially be used to treat meniscal tears or defects.

Transcriptome Profiling of Atlantic Salmon Adherent Head Kidney Leukocytes Reveals That Macrophages Are Selectively Enriched During Culture

The Atlantic salmon (Salmo salar) is an economically important fish, both in aquaculture and in the wild. In vertebrates, macrophages are some of the first cell types to respond to pathogen infection and disease. While macrophage biology has been characterized in mammals, less is known in fish. Our previous work identified changes in the morphology, phagocytic ability, and miRNA profile of Atlantic salmon adherent head kidney leukocytes (HKLs) from predominantly “monocyte-like” at Day 1 of in vitro culture to predominantly “macrophage-like” at Day 5 of culture. Therefore, to further characterize these two cell populations, we examined the mRNA transcriptome profile in Day 1 and Day 5 HKLs using a 44K oligonucleotide microarray. Large changes in the transcriptome were revealed, including changes in the expression of macrophage and immune-related transcripts (e.g. csf1r, arg1, tnfa, mx2), lipid-related transcripts (e.g. fasn, dhcr7, fabp6), and transcription factors involved in macrophage differentiation and function (e.g. klf2, klf9, irf7, irf8, stat1). The in silico target prediction analysis of differentially expressed genes (DEGs) using miRNAs known to change expression in Day 5 HKLs, followed by gene pathway enrichment analysis, supported that these miRNAs may be involved in macrophage maturation by targeting specific DEGs. Elucidating how immune cells, such as macrophages, develop and function is a key step in understanding the Atlantic salmon immune system. Overall, the results indicate that, without the addition of exogenous factors, the adherent HKL cell population differentiates in vitro to become macrophage-like.

Spontaneous Cell Detachment and Reattachment in Cancer Cell Lines: An In Vitro Model of Metastasis and Malignancy.

There is an unmet need for simplified in vitro models of malignancy and metastasis that facilitate fast, affordable and scalable gene and compound analysis. “Adherent” cancer cell lines frequently release “free-floating” cells into suspension that are viable and can reattach. This, in a simplistic way, mimics the metastatic process. We compared the gene expression profiles of naturally co-existing populations of floating and adherent cells in SW620 (colon), C33a (cervix) and HeLa (cervix) cancer cells. We found that 1227, 1367 and 1333 genes were at least 2-fold differentially expressed in the respective cell lines, of which 122 were shared among the three cell lines. As proof of principle, we focused on the anti-metastatic gene NM23-H1, which was downregulated both at the RNA and protein level in the floating cell populations of all three cell lines. Knockdown of NM23-H1 significantly increased the number of floating (and viable) cells, whereas overexpression of NM23-H1 significantly reduced the proportion of floating cells. Other potential regulators of these cellular states were identified through pathway analysis, including hypoxia, mTOR (mechanistic target of rapamycin), cell adhesion and cell polarity signal transduction pathways. Hypoxia, a condition linked to malignancy and metastasis, reduced NM23-H1 expression and significantly increased the number of free-floating cells. Inhibition of mTOR or Rho-associated protein kinase (ROCK) significantly increased cell death specifically in the floating and not the adherent cell population. In conclusion, our study suggests that dynamic subpopulations of free-floating and adherent cells is a useful model to screen and identify genes, drugs and pathways that regulate the process of cancer metastasis, such as cell detachment and anoikis.

Inflammatory Pre-Conditioning of Adipose-Derived Stem Cells with Cerebrospinal Fluid from Traumatic Brain Injury Patients Alters the Immunomodulatory Potential of ADSC Secretomes.

Immunomodulation by adipose-tissue-derived stem cells (ADSCs) is of special interest for the alleviation of damaging inflammatory responses in central nervous system injuries. The present study explored the effects of cerebrospinal fluid (CSF) from traumatic brain injury (TBI) patients on this immunomodulatory potential of ADSCs. CSF conditioning of ADSCs increased messenger RNA levels of both pro- and anti-inflammatory genes compared to controls. Exposure of phorbol-12-myristate-13-acetate-differentiated THP1 macrophages to the secretome of CSF-conditioned ADSCs downregulated both proinflammatory (cyclooxygenase-2, tumor necrosis factor alpha) and anti-inflammatory (suppressor of cytokine signaling 3, interleukin-1 receptor antagonist, and transforming growth factor beta) genes in these cells. Interleukin-10 expression was elevated in both naïve and conditioned secretomes. ADSC secretome treatment, further, induced macrophage maturation of THP1 cells and increased the percentage of CD11b+, CD14+, CD86+, and, to a lesser extent, CD206+ cells. This, moreover, enhanced the phagocytic activity of CD14+ and CD86+ cells, though independently of pre-conditioning. Secretome exposure, finally, also induced a reduction in the percentage of CD192+ adherent cells in cultures of peripheral blood mononuclear cells (PBMCs) from both healthy subjects and TBI patients. This limited efficacy (of both naïve and pre-conditioned secretomes) suggests that the effects of lymphocyte-monocyte paracrine signaling on the fate of cultured PBMCs are strongest upon adherent cell populations.

Analysis of Same Selected Immunomodulatory Properties of Chorionic Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) represent a population of adherent cells that can be isolated from multiple adult tissues. MSCs have immunomodulatory capacity and the ability to differentiate into many cell lines. Research study examines the immunomodulatory properties of MSCs isolated from chorion (CMSCs). Following the stimulation process, it was found that MSCs are capable of immunomodulatory action via the release of bioactive molecules as well as through direct contact with the immune cells. Immunomodulatory potential of the CMSCs was analyzed by modifying proliferative capacity of mitogen-activated lymphocytes. CMSCs and lymphocytes were tested in cell-to-cell contact. Lymphocytes were stained with carboxyfluorescein diacetate succinimidyl ester. Inhibition of the proliferation of activated lymphocytes was observed. Following the co-cultivation, the expression of markers involved in the immune response modulation was assessed. Afterwards, an increase in CMSCs expression of IL-10 was detected. Following the co-cultivation with activated lymphocyte, adhesion molecules CD54 and CD44 in the CMSCs increased. An increase of CD54 expression was observed. The properties of CMSCs, adherence and differentiation ability, were confirmed. The phenotype of CMSCs CD105+, CD90+, CD73+, CD44+, CD29+, CD45−, CD34−, CD54+ was characterized. It was demonstrated that chorion-derived MSCs have important immunomodulatory effects.

Kinetic Measurement of Apoptosis and Immune Cell Killing Using Live-Cell Imaging and Analysis.

The rapid, efficient detection of cell death is critical for characterizing the underlying biology of in vitro disease models and, in particular, immunotherapy products used for preclinical therapeutic research. Traditional endpoint assays are laborious to perform for mass screening of therapeutic candidates and may fail to fully capture the kinetics of events surrounding the initiation, duration, and mechanisms of cell death-important events that may affect translational relevance and impact therapeutic decision-making during development. Here, we describe simple, efficient methods to measure apoptosis and immune cell killing in both adherent and nonadherent cell populations using the Incucyte® Live-Cell Analysis system and associated nonperturbing reagents, cells, and protocols. Assays are performed in the user's own incubator with minimal disturbance and may be readily incorporated into existing workflows. Users may multiplex to maximize data collection from each sample. The integrated, user-friendly software does not require advanced technical training, enabling rapid analysis. Taken together, this method provides essential kinetic insight for greater understanding of cell death and the dynamic interactions between immune cells and their targets.

Transcriptome Analysis Reveals High Similarities between Adult Human Cardiac Stem Cells and Neural Crest-Derived Stem Cells.

Simple SummaryThe regeneration of nearly all organs of the human body mainly depends on the functionality of adult stem cell populations that reside in their respective niches and can be activated upon injuries or other damages. These stem cell populations greatly differ in their expression profile of molecular markers, which greatly influences their potential use in regenerative medicine. Neural crest-derived stem cells are a prominent subpopulation of adult stem cells and are known for their high regenerative potential. Within this study, we compared two adult human stem cell populations, namely neural crest-derived inferior turbinate stem cells from the nasal cavity and human cardiac stem cells from the heart, using global gene expression profiling. Here, we found differences that correspond to the tissue sources of origin but also similarities in the expression of markers that are associated with the neural crest. Further classifying nasal stem cells and cardiac stem cells in a broader context, we identified clear similarities between both populations and other adherent stem cell populations compared to non-adherent progenitor cells of the blood system. The analyses provided here might help to understand the differences and similarities between different adult human stem cell populations.For the identification of a stem cell population, the comparison of transcriptome data enables the simultaneous analysis of tens of thousands of molecular markers and thus enables the precise distinction of even closely related populations. Here, we utilized global gene expression profiling to compare two adult human stem cell populations, namely neural crest-derived inferior turbinate stem cells (ITSCs) of the nasal cavity and human cardiac stem cells (hCSCs) from the heart auricle. We detected high similarities between the transcriptomes of both stem cell populations, particularly including a range of neural crest-associated genes. However, global gene expression likewise reflected differences between the stem cell populations with regard to their niches of origin. In a broader analysis, we further identified clear similarities between ITSCs, hCSCs and other adherent stem cell populations compared to non-adherent hematopoietic progenitor cells. In summary, our observations reveal high similarities between adult human cardiac stem cells and neural crest-derived stem cells from the nasal cavity, which include a shared relation to the neural crest. The analyses provided here may help to understand underlying molecular regulators determining differences between adult human stem cell populations.

In-situ proliferation contributes to the accumulation of myeloid cells in the spleen during progressive experimental visceral leishmaniasis.

Visceral leishmaniasis (VL) is characterized by expansion of myeloid cells in the liver and spleen, which leads to a severe splenomegaly associated with higher risk of mortality. This increased cellularity is thought to be a consequence of recruitment of cells to the viscera. We studied whether the local proliferation of splenic myeloid cells contributes to increased splenic cellularity. We found that a monocyte-like population of adherent splenic cells from Leishmania donovani-infected hamsters had enhanced replicative capacity ex vivo and in vivo (BrdU incorporation, p<0.0001). In vitro assays demonstrated that proliferation was more pronounced in the proinflammatory M1 environment and that intracellular infection prevented proliferation. Secondary analysis of the published splenic transcriptome in the hamster model of progressive VL revealed a gene expression signature that included division of tumoral cells (Z = 2.0), cell cycle progression (Z = 2.3), hematopoiesis (Z = 2.8), proliferation of stem cells (Z = 2.5) and overexpression of proto-oncogenes. Regulators of myeloid cell proliferation were predicted in-silico (CSF2, TLR4, IFNG, IL-6, IL-4, RTK signaling, and STAT3). The in-silico prediction was confirmed with chemical inhibitors of PI3K/AKT, MAPK and STAT3 which decreased splenic myeloid cell division ex vivo. Hamsters infected with L. donovani treated with a STAT3 inhibitor had reduced in situ splenic myeloid proliferation (p = 0.03) and parasite burden. We conclude that monocyte-like myeloid cells have increased STAT3-dependent proliferation in the spleen of hamsters with visceral leishmaniasis and that inhibition of STAT3 reduces myeloid cell proliferation and parasite burden.

Adherent Intestinal Cells From Atlantic Salmon Show Phagocytic Ability and Express Macrophage-Specific Genes.

Our knowledge of the intestinal immune system of fish is rather limited compared to mammals. Very little is known about the immune cells including the phagocytic cells in fish intestine. Hence, employing imaging flow cytometry and RNA sequencing, we studied adherent cells isolated from healthy Atlantic salmon. Phagocytic activity and selected gene expression of adherent cells from the distal intestine (adherent intestinal cells, or AIC) were compared with those from head kidney (adherent kidney cells, or AKC). Phagocytic activity of the two cell types was assessed based on the uptake of Escherichia coli BioParticlesTM. AIC showed phagocytic ability but the phagocytes were of different morphology compared to AKC. Transcriptomic analysis revealed that AIC expressed genes associated with macrophages, T cells, and endothelial cells. Heatmap analysis of selected genes indicated that the adherent cells from the two organs had apparently higher expression of macrophage-related genes. We believe that the adherent intestinal cells have phagocytic characteristics and high expression of genes commonly associated with macrophages. We envisage the possibilities for future studies on enriched populations of adherent intestinal cells.

Quantifying the Rate, Degree, and Heterogeneity of Morphological Change during an Epithelial to Mesenchymal Transition Using Digital Holographic Cytometry

Cells in complex organisms can transition between epithelial and mesenchymal phenotypes during both normal and malignant physiological events. These two phenotypes are not binary, but rather describe a spectrum of cell states along an axis. Mammalian cells can undergo dynamic and heterogenous bidirectional interconversions along the epithelial–mesenchymal phenotypic (EMP) spectrum, and such transitions are marked by morphological change. Here, we exploit digital holographic cytometry (DHC) to develop a tractable method for monitoring the degree, kinetics, and heterogeneity of epithelial and mesenchymal phenotypes in adherent mammalian cell populations. First, we demonstrate that the epithelial and mesenchymal states of the same cell line present distinct DHC-derived morphological features. Second, we identify quantitative changes in these features that occur hours after induction of the epithelial to mesenchymal transition (EMT). We apply this approach to achieve label-free tracking of the degree and the rate of EMP transitions. We conclude that DHC is an efficient method to investigate morphological changes during transitions between epithelial and mesenchymal states.

Serum-free cryoprotectant reagent: Cell Rev™ HQ-Store, improves cell recovery rate and other intrinsic cell properties including MSC self-renewal and differentiation potentials

Background & Aim Cellular cryopreservation is a linchpin of cell therapy and manufacturing. Without proper cryoprotectant reagents, the quality of cellular properties such as basic cell viability, cell attachment, growth, differentiation, characters or even cell death could be jeopardized. The use of fetal bovine serum (FBS) in cell freezing reagents have grown to be laboratory standard practice. However, the composition of the FBS is not well defined both qualitative and quantitatively and also have batch- to – batch variations and most importantly is a loophole subjected to potential contamination of cell cultures. From the biosafety aspects, sera may contain harmful factors like mycoplasma, endotoxin, viral contaminants or prions which increase the probability of the transmission of potentially fatal diseases to human patients. Methods, Results & Conclusion To minimize the possible risks, we have developed a totally chemically defined cryoprotectant recipe to preserve the important qualities of the cells particularly mesenchymal Stem Cells (MSCs). In this study, Mesenchymal Stem Cells have been cryopreserved as adherent cell populations in widely used cryoprotectant recipe, 20% FBS 10% dimethyl sulfoxide (DMSO) and Cell Rev™ HQ-Store defined serum-free cell freezing reagents. Our serum-free cryopreservation recipe has shown a significantly improved post-thawed recovery rate of cells to 95%. Interestingly, MSCs that have been stored in our defined cryoprotectant showed more than 50% enhanced n self-renewal while retaining differentiation potentials. Most importantly, these high-quality cryopreservation remains consistent with more than 10 freeze-thaw cycles, and longer than 2 yrs extended storage. These data illustrate that cell freezing solutions affect not only cytotoxicity but also critical properties of cellular proliferation and differentiation.

Flow cytometry and live cell imaging for characterization of cell surface markers on human tissue-derived progenitors

Background & Aim Podoplanin (PDPN) was recently reported as a valuable potential marker for identifying human skeletal stem cells (SSCs). These are a subset of connective tissue progenitors (CTPs), a highly variable, and tissue specific group of cells capable of clonal propagation, proliferation and differentiation in vitro. Fluorescence-activated cell sorting (FACS) is a power tool for parsing cell markers, but FACS has a negative effect on colony forming efficiency. Development of better methods for isolating SSCs from human tissues using cell markers is a key step toward more viable methods of cell expansion. Our team has developed methods of live-cell large field of view (LFOV) imaging techniques that enable the identification and characterization of colony founding CTPs in culture soon after isolation and prior to cell division. The goal of this project is to define and validate the sensitivity and specificity of two LFOV imaging systems to detect the surface marker PDPN in comparison to flow cytometry. Methods, Results & Conclusion Bone marrow was aspirated from the iliac crest of consented donors. Nucleated cells were isolated. Live staining methods using anti-PDPN antibody, tested on positive control cell line U87 and negative control Jurkat, were defined for bone marrow-derived cells in suspension (Figure 1). The Cell X™ automation platform was used (Cell X Technologies) and the EVOS FL Auto 2 microscope (ThermoFisher Scientific) were used to capture LFOV images at 2 and 24 hours after plating. Subsequently, LFOV phase contrast images were collected every hour for 10 days, until colonies formed. Videos were reversed to identify individual colony founding CTPs. Quantitative image analysis was used to overlay areas of anti-PDPN and anti-CD45 staining with colony founding cells to determine the prevalence of PDPN+ and PDPN- cells among CTPs as well as the overall prevalence of PDPN+ cells in the entire adherent cell population as assessed using these methods. These data were then used to calculate the apparent prevalence of PDPN+ cells and the range of intensity of staining among CTPs, non-CTP adherent cells and total cells using each methods, with a parallel analysis using flow cytometry (LSR II). EVOS FL Auto 2 and Cell X devices are both able to detect PDPN+ and PDPN- cells in control cell lines (Figure 2) and among bone-marrow derived cells, enabling their use in further characterization of colony founding cell surface markers (CD146, CD271, HA).

Expansion microscopy for the analysis of centrioles and cilia.

SummaryCentrioles are vital cellular structures that organise centrosomes and cilia. Due to their subresolutional size, centriole ultrastructural features have been traditionally analysed by electron microscopy. Here we present an adaptation of magnified analysis of the proteome expansion microscopy method, to be used for a robust analysis of centriole number, duplication status, length, structural abnormalities and ciliation by conventional optical microscopes. The method allows the analysis of centriole's structural features from large populations of adherent and nonadherent cells and multiciliated cultures. We validate the method using EM and superresolution microscopy and show that it can be used as an affordable and reliable alternative to electron microscopy in the analysis of centrioles and cilia in various cell cultures.Lay DescriptionCentrioles are microtubule‐based structures organised as ninefold symmetrical cylinders which are, in human cells, ∼500 nm long and ∼230 nm wide. Centrioles assemble dozens of proteins around them forming centrosomes, which nucleate microtubules and organise spindle poles in mitosis. Centrioles, in addition, assemble cilia and flagella, two critically important organelles for signalling and motility. Due to centriole small size, electron microscopy has been a major imaging technique for the analysis of their ultrastructural features. However, being technically demanding, electron microscopy it is not easily available to the researchers and it is rarely used to collect large datasets. Expansion microscopy is an emerging approach in which biological specimens are embedded in a swellable polymer and isotopically expanded several fold. Physical separation of cellular structures allows the analysis of, otherwise unresolvable, structures by conventional optical microscopes. We present an adaptation of expansion microscopy approach, specifically developed for a robust analysis of centrioles and cilia. Our protocol can be used for the analysis of centriole number, duplication status, length, localisation of various centrosomal components and ciliation from large populations of cultured adherent and nonadherent cells and multiciliated cultures. We validate the method against electron microscopy and superresolution microscopy and demonstrate that it can be used as an accessible and reliable alternative to electron microscopy.