Cell Strainer Research Articles

Optimization of the method for isolation of epithelial cells from the non-glandular part of the rat stomach for flow cytometry

Traditional methods in cell proliferation studies are based on immunohistochemical detection of proliferating cells in the target tissue. Since they are time consuming, optimization of novel, more efficient methods is important for large scale proliferation studies. In this study, we aimed to optimize the isolation of single epithelial rat forestomach cells for flow cytometry. As a marker of cellular proliferation we used the Ki-67 antibody to detect this nuclear protein expressed in proliferating cells. We also performed immunohistochemical detection of Ki-67 positive cells and propidium iodide staining to validate the results. 3-tert- butyl -4-hydroxyanisole was used as the positive control to ensure cellular proliferation. The results showed that isolation of epithelial cells with collagenase, trypsin and cell strainer ensures great cell viability (>95%) and the purity of the samples. Flow cytometry and immunostaining with the Ki-67 antibody indicated that 3-tert- butyl-4-hydroxyanisole treatment leads to a significant increase in proliferation. A significant positive correlation was observed between the results obtained by immunohistochemistry and flow cytometry, but the flow cytometric data had a smaller measurement error, suggesting the equal sensitivity and greater accuracy of this method. Propidium iodide staining showed that the percentage of cells in the G2+S phase of the cell cycle correlated positively with the percentage of Ki-67 positive cells assessed by flow cytometry, indicating that Ki-67 positive cells reflect an active dividing cell pool. We conclude that the isolation of forestomach epithelial cells described is a simple and reliable method for obtaining viable cells for use in flow cytometry. Compared to immunohistochemistry, flow cytometric detection of the Ki-67 antigen is equally sensitive, but much faster and provides more accurate results.

EFFECT OF BOILING AND MICROWAVE COOKING ON NUTRITIONAL, ANTI-NUTRITIONAL AND TOXICITY OF WILD EDIBLE PLANTS OF NORTH-EASTERN REGION IN INDIA

Objective: The target of this appraisal was to explore the impacts of various cooking techniques, for example, boiling and microwave cooking on nutritional, antinutritional, minerals content and in vitro toxicity of ten wild consumable plants viz. Zanthoxylum acanthopodium, Viburnum foetidum, Houttuynia cordata, Sonchus arvensis, Oenanthe linearis, Perilla ocymoides, Clerodendrum colebrookeanum, Solanum gilo, Solanum kurzii, Potentilla lineata, widely consumed by the common individuals of North-Eastern area in India.
 Methods: The proximate parameters like ash, moisture, protein, fat, fibre, carbohydrate, energy content, minerals viz. sodium, calcium, potassium, iron, magnesium, manganese, copper, zinc and antinutritional parameters like oxalate, phytate, tannin, saponin, cyanogenic glycoside content were evaluated in the selected wild edible plants using standard food analysis techniques. In vitro haemolytic toxicity of aqueous extracts (100, 300 and 500µg/cc) of ten palatable wild plants was done with the blood samples were gathered from healthy rat, mixed with Ethylenediaminetetraacetic acid(EDTA) and centrifuged at 5,000 Revolutions Per Minute (rpm) for five minutes. The 10 % erythrocyte suspension was set up in sterile Phosphate buffer saline (PBS, pH 7.4) for haemolytic examination. The genotoxic potential of the concentrates were assessed by a single-cell gel electrophoresis comet test. Cytotoxicity studies were evaluated with fresh goat livers procured from the local market were perfused in PBS (pH 7.4) with collagenase and the liver was then minced in minute pieces and cells were isolated utilizing cell strainer.
 Results: Both cooking medications diminished the congregations of ash, fat, minerals, antinutritional parts and the destructive nature of the consumable plants while the carbohydrate and fiber substance were expanded. The protein focuses in the wild edibles were expanded fundamentally (P<0.05) in the range from 1.26 to 10.45% on microwave cooking while the indistinguishable were exhausted in the range out of 2.20 to11.55% on boiling treatment. The microwave cooking demonstrated lesser misfortunes in minerals in the consumable plants than those cooked by frothing. The microwave cooking also caused the colossal rot (P<0.05) of antinutritional parameters and damaging tendency to a more significant degree than the sputtering medications of the wild edibles.
 Conclusion: Therefore, the outcomes uncovered that microwaving of appealing plants could be prescribed to expand the supporting quality and to diminish the fat, threatening to dietary structure and lethality. The toxicity assessment of the consumable plants at cell and genomic level showed that these are harmless to consume.

DNA methylation modulates allograft survival and acute rejection after renal transplantation by regulating the mTOR pathway

Acute rejection (AR) can lead to allograft dysfunction following renal transplantation, despite immunosuppressive treatments. Accumulating evidence points out a role for epigenetic modification in immune responses. However, the mechanism and contribution of DNA methylation in allograft survival remain unclear. In this study, we followed up patients who successively experienced end‐stage renal disease, renal transplantation with allograft function or dysfunction, and hemodialysis. Peripheral blood mononuclear cells were collected at different time points for analysis of the DNA methylation. Epigenetic modifier analysis was also performed to explore its effect of methylation in a mouse model of AR. Compared with the allograft‐stable cohort, patients who experienced AR‐induced allograft dysfunction demonstrated more changes in methylation patterns. Pathway analysis revealed that the hypermethylated areas in the allograft dysfunction group were associated with genes related to the mechanistic target of rapamycin (mTOR) signaling pathway. Moreover, in the mouse AR model, treatment with the DNA methyltransferase inhibitor—decitabine regulated the Th1/2/17/regulatory T cell (Treg cell) immune response via its demethylating role in the suppressing the activity of the mTOR pathway, which ultimately ameliorated renal allograft‐related inflammatory injuries. These results revealed that changes in methylation accompany AR‐induced allograft dysfunction after renal transplantation. Epigenetics may provide new insights into predicting and improving allograft survival.

Efficient isolation of brain capillary from a single frozen mouse brain for protein expression analysis

Isolated brain capillaries are essential for analyzing the changes of protein expressions at the blood–brain barrier (BBB) under pathological conditions. The standard brain capillary isolation methods require the use of at least five mouse brains in order to obtain a sufficient amount and purity of brain capillaries. The purpose of this study was to establish a brain capillary isolation method from a single mouse brain for protein expression analysis. We successfully isolated brain capillaries from a single frozen mouse brain by using a bead homogenizer in the brain homogenization step and combination of cell strainers and glass beads in the purification step. Western blot and proteomic analysis showed that proteins expressed at the BBB in mouse brain capillaries isolated by the developed method were more enriched than those isolated from a pool of five mouse brains by the standard method. By using the developed method, we further verified the changes in expression of BBB proteins in Glut1-deficient mouse. The developed method is useful for the analysis of various mice models with low numbers and enables us to understand, in more detail, the physiology and pathology of BBB.

Isolation of Highly Migratory and Invasive Cells in Three-Dimensional Gels.

We developed a modified invasion assay in three-dimensional (3D) gels that permits isolation of invading cells as living cells, termed an invading cell-trapping (iCT) assay. A small cell strainer consisting of nylon mesh with pores of 40-µm square is used in this assay. A layer of gel composed of extracellular-matrix components is formed on each side of the nylon mesh, which permits cell migration or invasion from one gel layer to the other. At the end of the assay, the two gel layers are removed from the apparatus and easily separated from each other. Invading cells from the primary gel are trapped in the secondary gel, which maintains the morphology and other properties of the invasive cells in a 3D matrix. The cells that have invaded are observed and counted with a standard light microscope without cell staining. There is no need for a specialized microscope, imaging analysis software, or advanced cell-biological technical knowledge in this assay. This assay can also reduce measurement of nonspecific cell invasion by monitoring the upward invasion of cells. The viability of both invading and non-invading cells trapped in the gels can be assessed by typical colorimetric assays, if desired. This assessment characterizes the total number of cells (invading and non-invading cells) and the ratio of invading cells to total cells. By repeating the iCT assay, further enrichment of invasive and noninvasive cells can be attained. Thus, this assay improves comparative analyses between invasive and noninvasive cells. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Measuring upward cell invasion into collagen gel Basic Protocol 2: Measuring cell invasion from Matrigel into collagen gel Basic Protocol 3: Isolation and enrichment of highly invasive cells.

Extracellular vesicles derived from injured vascular tissue promote the formation of tertiary lymphoid structures in vascular allografts.

Tertiary lymphoid structures (TLS) accumulate at sites of chronic injury where they function as an ectopic germinal center, fostering local autoimmune responses. Vascular injury leads to the release of endothelial‐derived apoptotic exosome‐like vesicles (ApoExo) that contribute to rejection in transplanted organs. The purpose of the study was to evaluate the impact of ApoExo on TLS formation in a model of vascular allograft rejection. Mice transplanted with an allogeneic aortic transplant were injected with ApoExo. The formation of TLS was significantly increased by ApoExo injection along with vascular remodeling and increased levels of antinuclear antibodies and anti‐perlecan/LG3 autoantibodies. ApoExo also enhanced allograft infiltration by γδT17 cells. Recipients deficient in γδT cells showed reduced TLS formation and lower autoantibodies levels following ApoExo injection. ApoExo are characterized by proteasome activity, which can be blocked by bortezomib. Bortezomib treated ApoExo reduced the recruitment of γδT17 cells to the allograft, lowered TLS formation, and reduced autoantibody production. This study identifies vascular injury‐derived extracellular vesicles (ApoExo), as initiators of TLS formation and demonstrates the pivotal role of γδT17 in coordinating TLS formation and autoantibody production. Finally, our results suggest proteasome inhibition with bortezomib as a potential option for controlling TLS formation in rejected allografts.

Resveratrol Effect on Adipose-Derived Stem Cells Differentiation to Chondrocyte in Three-Dimensional Culture.

Purpose: Adipose stem cells (ASCs) are pluripotent cells with the ability of self-renewal and differentiation into different types of mesenchymal cells. As cartilage repair is difficult due to lack of blood capillary, resveratrol (Res) is a polyphenolic compound with diverse biological properties to be possibly used in this case. The aim of the present study was to investigate the effect of Res on differentiation of ASCs into chondrocyte in a three-dimensional (3D) culture model. Methods: Subcutaneous adipose tissues were prepared and digested enzymatically, and passed through cell strainer. ASCs were harvested in the fourth passage, and divided into five groups. The control group received chondrogenic differentiation medium (CDM) while the experimental groups received CDM plus different doses of Res (1, 10, 20, and 50 µM) for 21 days. Expression of cartilage specific genes and Sirtuin1 (SIRT 1), cell viability, apoptosis and ferric reducing antioxidant power (FRAP) were detected using reverse transcription polymerase chain reaction (RT-PCR), MTT assay, TUNEL and acridine orange/ethidium bromide (AO/EB) staining. One-way ANOVA and non-parametric Mann-Whitney U test were used for data analyses. Results: ASCs were differentiated to chondrocyte by CDM in a three-dimensional culture. 10 and 20 µM doses of Res showed the most proliferating effect on ADSCs. The SIRT 1 genes expression and FRAP level also increased significantly compared to the control group (P<0.05). Also, OD of cell increased whereas apoptosis decreased. Conclusion: 3D culture was a suitable condition for ASCs differentiation to chondrocyte, and lower doses of Res exert proliferation effect on ASCs.

Sonic Hedgehog, a Novel Endogenous Damage Signal, Activates Multiple Beneficial Functions of Human Endometrial Stem Cells.

Local endometrial stem cells play an important role in regulating endometrial thickness, which is an essential factor for successful embryo implantation and pregnancy outcomes. Importantly, defects in endometrial stem cell function can be responsible for thin endometrium and subsequent recurrent pregnancy losses. Therefore, many researchers have directed their efforts toward finding a novel stimulatory factor that can enhance the regenerative capacity of endometrial stem cells. Sonic hedgehog (SHH) is a morphogen that plays a key role in regulating pattern formation throughout embryonic limb development. In addition to this canonical function, we identified for the first time that SHH is actively secreted as a stem cell-activating factor in response to tissue injury and subsequently stimulates tissue regeneration by promoting various beneficial functions of endometrial stem cells. Our results also showed that SHH exerts stimulatory effects on endometrial stem cells via the FAK/ERK1/2 and/or phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways. More importantly, we also observed that endometrial stem cells stimulated with SHH showed markedly enhanced differentiation and migratory capacities and subsequent invivo therapeutic effects in an endometrial ablation animal model.

Optimization of The Cell Aggregates Method for Isolation and Purification of Human Granulosa Cells from Follicular Fluid.

BackgroundAspirated ovarian follicular fluids (FF) contain luteal granulosa cells (LGCs) and other contaminating cell types. Several strategies, such as the antibody binding methods, the flask method, the cell strainer and positive selection of granulosa aggregates after density gradient (DG) centrifugation, were tested as LGC purification methods. Each of these strategies has its own advantages and disadvantages. Positive selection of granulosa aggregates after DG centrifugation is simple, rapid and efficient in terms of LGC recovery. However, it results in a low purity. Here, we aimed to test whether modifying the traditional protocol by collecting the aggregates from the FF, before the DG centrifugation could decrease the percentage of contaminating cells.Materials and MethodsIn the present prospective study, 32 FF, from 32 women,were randomly assigned into one of the two purification techniques: positive selection of granulosa aggregates from the FF, after DG centrifugation (DG/ Agg, n=16) or positive selection of granulosa aggregates from the FF, before DG centrifugation (Agg/DG, n=16). At the end of each procedure cell count, vitality, morphology and purity of the cell suspension were evaluated.ResultsNo significant difference was detected in the total number of GCs between DG/Agg and Agg/DG (P>0.05). However, higher percentage of GCs with normal morphology was detected in Agg/DG compared to DG/Agg (P<0.001). Moreover, lower percentages of white blood cells (P<0.01), red blood cells (P<0.001) and epithelial cells (P<0.01) were identified in Agg/DG compared to DG/Agg.ConclusionHere we showed that positive selection of granulosa aggregates from the FF prior to DG technique had a higher purity compared to the traditional protocol. Thus, it could be a method of choice to prepare GCs for research purposes in clinical in vitro fertilization settings.

Neurokinin-1 Receptor Antagonism Ameliorates Dry Eye Disease by Inhibiting Antigen-Presenting Cell Maturation and T Helper 17 Cell Activation

Neuroinflammation plays an important role in the pathogenesis of ocular surface disease, including dry eye disease (DED), but little is known about the contribution of substance P (SP) to DED. In this study, we investigated the expression of SP at the ocular surface and evaluated its effect on maturation of antigen-presenting cells (APCs), the key cell component involved in the induction of type 17 helper T-cell (Th17) response in DED. The effect of topical blockade of SP signaling was further investigated using neurokinin-1 receptor (NK1R) inhibitors on APC maturation, Th17 cell activation, and disease severity in a mouse model of DED. The results demonstrate that SP is constitutively expressed at the ocular surface, and trigeminal ganglion neurons are the major source of SP in DED. SP derived from trigeminal ganglion enhanced the expression of major histocompatibility complex class II maturation marker by bone marrow-derived dendritic cells, an effect that is abrogated by blockade of SP signaling using NK1R antagonist spantide. Finally, using a well-established murine model of DED, topical treatment of DED mice with NK1R antagonists CP-99,994 and L-733,060 suppressed APC acquisition of major histocompatibility complex class II, reduced Th17 cell activity, and ameliorated DED severity. These findings are of translational value, as they suggest that antagonizing NK1R-mediated SP signaling may be an effective strategy in suppressing Th17-mediated ocular surface disease.

A simple and highly purified method for isolation of glomeruli from the mouse kidney.

Highly purified mouse glomeruli are of great value for studying glomerulus-associated kidney diseases. Here, we developed a simple and rapid procedure for mouse glomerular isolation with large quantity and high purity based on the combination of size-selective sieving and differential adhesion techniques, which we termed the "differential adhesion method." In this method, mouse renal cortices were minced and digested with collagenase. Glomeruli were disassociated from tubules by successive sieving through 105-, 75-, and 40-μm cell strainers. The retained glomeruli-rich preparation on the 40-μm strainer was rinsed into a cell culture dish to allow tubules to adhere quickly to the dish while leaving most glomeruli floating (termed "differential adhesion"). The floating glomerular fraction was then subjected to another wash through the 40-μm strainer followed by an additional differential adhesion step to obtain highly purified glomeruli with yields of 8,357 ± 575 and purity of 96.1 ± 1.8% from one adult C57BL/6 mouse. The purity of the isolated glomeruli was further confirmed by high expression of the podocyte marker nephrin without detectable tubular marker cadherin-16. Importantly, we also found that although both the quantity and purity of the isolated glomeruli by this and the established Dynabeads method were comparable, glomeruli isolated by the current method showed much less inflammatory stress in terms of proinflammatory cytokine expression than the Dynabeads method. In conclusion, we established a newly mouse glomerular isolation method that is simple, rapid, cost effective, and productive. It provides an advanced methodology for research into glomerulus-related kidney diseases in the mouse.

A step towards enzyme-free tissue dissociation

Abstract The future of personalized diagnostics commences on the single cell level. Even high-end technologies like Next Generation Sequencing can be improved if applied on pure single cell populations (e.g., tumor cells without contaminating stromal cells) or on a single cell level (DNA/RNA sequencing). The vast majority of these technologies need individual and preferably undistorted cells for the analytical process. Thus, decisive prerequisite for high-end analytics is to remove cells from their tissue matrix as gently as possible. This can be accomplished by an enzyme-free, fast and reproducible approach of generating pure and individual single cells from tissue samples. In this study we demonstrate the utility of a semi-automated Tissue Grinder that is compatible with standard 50 ml centrifuge tubes and standard cell strainer for mechanically, nonenzymatic and parallel processing of tissue samples. We show that without enzymatic treatment viable single-cell yields match or exceed reference enzymatic methods, while reducing processing time by at least 80%.

Measurement of DNA damage by CellProfiler software in the liver of Caspian white fish exposed to environmental concentrations of fipronil

Pesticides are one of the most important factors threatening the ecosystem of the Caspian Sea. Due to prevalence of gastrointestinal cancer in Northern provinces of Iran and the possible involvement of pesticides in this case, genetic toxicity of fipronil insecticide on the liver of the Caspian white fish (Rutilus kutum) was studied. Twelve Caspian whitefish fingerlings (five groups including control and treatment groups) were exposed to environmental concentrations of fipronil (0.1, 1, 5 and 10 µg/L) to evaluate the effects of this pesticide on the DNA of fish liver cells. After 14 days of exposure, liver samples were taken. The samples were converted to separated cells after mincing, centrifugation and filtering through the cell strainer, and then dispersed on the slides coated with low melting agarose. After electrophoresis, the slides were stained with SYBRGreen. CellProfiler software was used to analyze the comets. The results showed that increasing the environmental concentration of fipronil increases the damage to DNA. In general, this study showed that fipronil has the potential of damage to DNA and can be considered as one of the factors reducing the survival rate of Caspian white fish fingerling and larva, and possibly expanding the cancer in Northern provinces of Iran.

IL-6 and IL-8 secreted by tumour cells impair the function of NK cells via the STAT3 pathway in oesophageal squamous cell carcinoma

BackgroundRecurrence and metastasis are the leading causes of tumour-related death in patients with oesophageal squamous cell carcinoma (ESCC). Tumour-infiltrating natural killer cells (NK cells) display powerful cytotoxicity to tumour cells and play a pivotal role in tumour therapy. However, the phenotype and functional regulation of NK cells in oesophageal squamous cell carcinoma (ESCC) remains largely unknown.MethodsSingle cell suspensions from blood and tissue samples were isolated by physical dissociation and filtering through a 70 μm cell strainer. Flow cytometry was applied to profile the activity and function of NK cells, and an antibody chip experiment was used to identify and quantitate cytokine levels. We studied IL-6 and IL-8 function in primary oesophageal squamous carcinoma and NK cell co-cultures in vitro and by a xenograft tumour model in vivo. Western blotting was used to quantitate STAT3 (signal transducer and activator of transcription 3) and p-STAT3 levels. Finally, we performed an IHC array to analyse IL-6/IL-8 (interleukin 6/interleukin 8) expression in 103 pairs of tumours and matched adjacent tissues of patients with ESCC to elucidate the correlation between IL-6 or IL-8 and clinical characteristics.ResultsThe percentages of NK cells in both peripheral blood and tumour tissues from patients with ESCC were significantly increased in comparison with those in the controls and correlated with the clinical characteristics. Furthermore, the decrease in activating receptors and increase in inhibitory receptors on the surface of tumour-infiltrating NK cells was confirmed by flow cytometry. The level of granzyme B, the effector molecule of tumour-infiltrating NK cells, was also decreased. Mechanistically, primary ESCC cells activated the STAT3 signalling pathway on NK cells through IL-6 and IL-8 secretion, leading to the downregulation of activating receptors (NKp30 and NKG2D) on the surface of NK cells. An ex vivo study showed that blockade of STAT3 attenuated the IL-6/IL-8-mediated impairment of NK cell function. Moreover, the expression of IL-6 or IL-8 in tumour tissues was validated by immunohistochemistry to be positively correlated with tumour progression and poor survival, respectively.ConclusionsTumour cell-secreted IL-6 and IL-8 impair the activity and function of NK cells via STAT3 signalling and contribute to oesophageal squamous cell carcinoma malignancy.

De novo generation of a functional human thymus from induced pluripotent stem cells

A proper functional thymus is required for generation of T cell–mediated immunity. This is dramatically illustrated by patients lacking a thymus, such as children with complete DiGeorge syndrome, which is fatal if left untreated.1-3 Therapeutic options for such patients are limited and confined to transplantation of small fragments from allogeneic neonatal thymi.

Straightforward method for singularized and region-specific CNS microvessels isolation

BackgroundCurrent methods for murine brain microvasculature isolation requires the pooling of brain cortices while disregarding the rest of the CNS, making the analysis of single individuals non feasible. New methodEfficient isolation of brain microvessels requires the elimination of meninges, vessels of high caliber vessels and choroid plexus, commonly done by rolling the over filter paper, but can’t be done on other CNS regions. We overcome this hurdle by using a double-pronged pick, as well as elution and filtration through cell strainers after centrifugation. ResultsWe were able to develop a region-specific murine CNS microvessels isolation, that allows for the comparison of the neurovascular unit from these regions both within the same individual and between multiple individuals and/or treatment groups without pooling. Additionally, we were able to adapt this method to macaque CNS tissue. Comparison with existing method(s)Although similar to a previously published method that requires no enzymatic dissociation and no ultracentrifugation, it does differ in its ability to isolate from a single experimental animal and from non-cortical tissues. However, it relies heavily on the researcher dissecting skills and careful elution and filtration of re-suspended samples. ConclusionsCNS region-specific microvessels comparison can inform of molecular and/or cellular differences that would otherwise be obscured by excluding non-cortical tissue. Additionally, it allows for the unmasking of variations between individuals that remained hidden when pooling of multiple samples is the norm. Lastly, isolation of region-specific microvessels for non-human primate CNS allows for more translationally relevant studies of the BBB.

Cartilage Induction from Mouse Mesenchymal Stem Cells in High-density Micromass Culture.

Mesenchymal stem cells have the ability to differentiate into multiple lineages, including adipocytes, osteoblasts and chondrocytes. Mesenchymal stem cells can be induced to differentiate into chondrocytes in extracellular matrices, such as alginate or collagen gel. Mesenchymal stem cells in a cell pellet or micromass culture can be also induced to form cartilages in a defined medium containing chondrogenic cytokines, such as transforming growth factor-β (TGF-β). Here, we describe a simple method to form cartilage by seeding mesenchymal cells derived from limb-bud cells at high cell density. First, we dissected the limb buds from embryonic mice (embryonic day 12.5) and digested them with enzymes (dispase and collagenase). After filtration using a cell strainer, we seeded the cells at high density. Unlike other methods, the method described here is simple and does not require the use of specialized equipment, expensive materials or complex reagents.

One method to establish Epstein-Barr virus-associated NK/T cell lymphoma mouse models.

Novel nude mice model of human NK/T cell lymphoma were established by subcutaneously injecting two NK/T cell lymphoma cell lines into the right axillary region of mice and successful passages were completed by injecting cell suspension which was obtained through a 70‐μm cell strainer. These mice models and corresponding cell clones have been successfully developed for more than 8 generations. The survival rates of both resuscitation and transplantation in NKYS and YT models were 90% and 70% correspondingly. Pathologically, the tumour cells in all passages of the lymphoma‐bearing mice and cell lines obtained from tumours were parallel to initial cell lines. Immunologically, the tumour cells expressed the characteristics of the primary and essential NK/T lymphomas. The novel mice models maintained the essential features of human NK/T cell lymphoma, and they would be ideal tools in vivo for further research of human NK/T cell lymphoma.

Isolation of cancer cells with augmented spheroid-forming capability using a novel tool equipped with removable filter.

Three-dimensional (3D) cell culture systems have been used to obtain multicellular spheroidal cell aggregates, or spheroids, from cancer cells. However, it is difficult to efficiently prepare large tumor-derived spheroids from cancer cells. To circumvent this problem, we here used a tool equipped with removal membrane, called Spheroid Catch, for the selection and enrichment of large-sized and/or size-matched spheroids from human squamous cell carcinoma (SAS cells) without loss of recovery. After a five-round process of selection and enrichment, we successfully isolated a subpopulation of SAS cells with augmented spheroid-forming capability, named eSAS: the efficiency of spheroid formation is 28.5% (eSAS) vs 16.8% (parental SAS). Notably, we found that some of eSAS cells survived after exposure of high doses of cisplatin in 3D culture. Moreover, orthotopic implantation by injecting eSAS cells into the tongues of nude mice showed reduced survival rate and increased tumor growth compared with those of nude mice injected with SAS cells. These results suggest that spheroids exhibiting properties of higher spheroid forming capacity can be efficiently collected by using Spheroid Catch. Indeed, genome-wide cDNA microarray and western blot analyses demonstrated higher mRNA and protein levels of hedgehog acyltransferase (HHAT), which is associated with stem maintenance in cell carcinoma by catalysing the N-palmitoylation of Hedgehog proteins, in eSAS cells than in SAS cells. We propose that Spheroid Catch could be useful for the study of spheroids, and potentially organoids, in the basic and clinical sciences, as an alternative method to other type of cell strainers.

Simple and cost-effective assay for isolating invasive living cells.

A modified invasion assay using a three-dimensional collagen gel was developed that enables isolation of invasive living cells; it was named the invading cell trapping (iCT) assay. A small cell strainer consisting of a nylon mesh with 40-μm2 pores was used, and collagen gel layers formed across the membrane. Test cells were seeded in the lower gel layer and invasive cells were attracted upward and trapped in the upper gel. After incubation, the collagen gel layers in cell strainers were easily separated and living cells in the gel were counted and analyzed. An advantage of the iCT assay is that it can capture living invasive cells in the upper gel while leaving noninvasive ones in the lower layer. Further enrichment of the two cell populations can be achieved by repeating the assay. Thus, the iCT assay allows comparative analysis of invasive versus noninvasive cells.