Cell Strainer Research Articles

Genome-wide screening for genes involved in the epigenetic basis of fragile X syndrome.

SummaryFragile X syndrome (FXS), the most prevalent heritable form of intellectual disability, is caused by the transcriptional silencing of the FMR1 gene. The epigenetic factors responsible for FMR1 inactivation are largely unknown. Here, we initially demonstrated the feasibility of FMR1 reactivation by targeting a single epigenetic factor, DNMT1. Next, we established a model system for FMR1 silencing using a construct containing the FXS-related mutation upstream to a reporter gene. This construct was methylated in vitro and introduced into a genome-wide loss-of-function (LOF) library established in haploid human pluripotent stem cells (PSCs), allowing the identification of genes whose functional loss reversed the methylation-induced silencing of the FMR1 reporter. Selected candidate genes were further analyzed in haploid- and FXS-patient-derived PSCs, highlighting the epigenetic and metabolic pathways involved in FMR1 regulation. Our work sheds light on the mechanisms responsible for CGG-expansion-mediated FMR1 inactivation and offers novel targets for therapeutic FMR1 reactivation.

OR26. Dedifferentiated Fat Cells versus Adipose Derived Mesenchymal Stem Cells Experimental Study about Adipose-derived Stem Cells for Cardiomyocyte Precursor using Traditional and Insert Culture

Abstract Background and Aims The discovery of dedifferentiated fat cell (DFAT) raises the question, are AMSC and DFAT the same type of mesenchymal stem cell (MSC)? We aim to analyze the immunocytochemistry (ICC) profile of both cells and the correlation between different methods with different passage to GATA-4 and cTnT expression in vitro. Methods and Results Human subcutaneous-fat tissue was isolated and divided into, first group, using single filtration by 100 µm cell strainer and second group using double filtration with addition of 70 µm strainer after the 100 µm. Cell obtained from both groups were labeled AMSC and DFAT respectively. Prior to passaging, CD34, CD45, CD90, and CD105 were analyze by ICC. After passaging, passage 1 and 4 was exposed to Gibco PSC Cardiomyocyte Differentiation Kit for 21 days. Colonies from days 7, -14, and -21 were analyze by ICC for GATA-4 and cTnT expression. Post hoc analysis shown that there is no significance difference in MSC markers expression between groups. There were significance differences in GATA-4 expression on all observation days, which decreased significantly from the 14th to the 21st day. Statistic significancy also found on cTnT expression, with a significant increase from day 14 to day 21. The filter variable consistently has a significant effect on the quantity of GATA-4 and cTnT expressions. Conclusion The study shows that different culture methods, presumably the use of filters, will have a significant effect on the differentiation of AMSC and DFAT into cardiomyocytes.

Easy to build cost-effective acute brain slice incubation system for parallel analysis of multiple treatment conditions

BackgroundAcute brain slices represent a powerful tool for analysis of brain function in physiology and pathology. Commercial systems and custom-build solutions with carbogen (95% O2/5% CO2) aeration, but they are expensive, have a high working volume requiring large amount of substances, and only limited options for treatment in parallel are possible. New methodWe developed a novel cost-effective incubation system using materials available in every laboratory, allowing parallel incubation of several treatment conditions, thus also reducing the number of experimental animals. Our system incubation parameters were optimized for cortical neuron observation. ResultsWe tested several different options using 6, 12 or 24 standard culture well plates, combining them with cell strainer baskets inside. The system was placed in a pre-warmed incubator at 37 °C. Carbogen was injected through a 22 gauge needle, positioned between the basket and the wall of the well. Best results were achieved in a 6-well plate. In 12 and 24-well plates bubbles accumulated beneath the basket, displacing it upwards, making it unsuitable for our purposes. The gas oxygenized the medium without mechanically disturbing the slices, protected within the strainer basket, but still allowing optimal diffusion through the 100 µm pores. In a 6-well plate, six simultaneous treatments were possible in parallel. LDH/Cytotoxicity tests showed an acute toxicity of less than 7%. The system lost about 2.5% per hour of the fluid through evaporation, which was replenished every 2 h. Up to 6 h after treatment, however, this evaporation was excellently tolerated by the neurons even without fluid replenishment, most probably due to the anti-swelling effect of the mildly hypertonic medium. We performed two staining procedures, working excellently with this experimental setup, namely – a modified DiI staining and a slice silver impregnation method, both confirming the intact neuronal morphology. Preserved CA3 calcium influx and removal response following KCl depolarization confirmed the normal physiology of the pyramidal neurons 6 h after exposure in the system. Comparison to existing methodsThe proposed system is much cheaper than the commercial solutions, can be constructed in any lab, allows up to 6 different treatments in parallel, which none of the existing systems allows. Antibiotic presence in the incubation medium and adequate evaporation control is required if longer incubation (> 6 h) is needed. Lower incubation volumes (3–6 ml) allow sparing expensive reagents. Our procedure was optimized for cortical neurons, further fine tuning to meet other specific requirements is possible. ConclusionsThe system we propose allows filling the gap for budget solutions for short to mid-term incubation of acute brain slices.

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Methionine deficiency and its hydroxy analogue influence chicken intestinal 3-dimensional organoid development.

Methionine and its hydroxy analogue (MHA) have been shown to benefit mouse intestinal regeneration. The intestinal organoid is a good model that directly reflects the impact of certain nutrients or chemicals on intestinal development. Here, we aimed to establish a chicken intestinal organoid culture method first and then use the model to explore the influence of methionine deficiency and MHA on intestinal organoid development. The results showed that 125-μm cell strainer exhibited the highest efficiency for chicken embryo crypt harvesting. We found that transforming growth factor-β inhibitor (A8301) supplementation promoted enterocyte differentiation at the expense of the proliferation of intestinal stem cells (ISC). The mitogen-activated protein kinase p38 inhibitor (SB202190) promoted intestinal organoid formation and enterocyte differentiation but suppressed the differentiation of enteroendocrine cells, goblet cells and Paneth cells. However, the suppression of enteroendocrine cell and Paneth cell differentiation by SB202190 was alleviated at the presence of A8301. The glycogen synthase kinase 3 inhibitor (CHIR99021), valproic acid (VPA) alone and their combination promoted chicken intestinal organoid formation and enterocyte differentiation at the expense of the expression of Paneth cells and goblet cells. Chicken serum significantly improved organoid formation, especially in the presence of A8301, SB202190, CHIR99021, and VPA, but inhibited the differentiation of Paneth cells and enteroendocrine cells. Chicken serum at a concentration of 0.25% meets the requirement of chicken intestinal organoid development, and the beneficial effect of chicken serum on chicken intestinal organoid culture could not be replaced by fetal bovine serum and insulin-like growth factor-1. Moreover, commercial mouse organoid culture medium supplemented with A8301, SB202190, CHIR99021, VPA, and chicken serum promotes chicken organoid budding. Based on the chicken intestinal organoid model, we found that methionine deficiency mimicked by cycloleucine suppressed organoid formation and organoid size, and this effect was reinforced with increased cycloleucine concentrations. Methionine hydroxy analogue promoted regeneration of ISC but decreased cell differentiation compared with the results obtained with L-methionine. In conclusion, our results provide a potentially excellent guideline for chicken intestinal organoid culture and insights into methionine function in crypt development.

Isolation and Differentiation of Adipose-Derived Stem Cells into Odontoblast-Like Cells: A Preliminary In Vitro Study.

Objective The aim of present study was to isolate and differentiate human adipose-derived stem cells (ASCs) into odontoblast-like cells. Materials and Methods In this experimental study, human adipose tissues were taken from the buccal fat pad of three individuals (mean age: 24.6 ± 2.1 years). The tissues were transferred to a laboratory in a sterile culture medium, divided into small pieces and digested by collagenase I (2 mg/mL, 60-90 minutes). ASCs were isolated by passing the cell suspension through cell strainers (70 and 40 µm), followed by incubation at 37ºC and 5% CO2in Dulbecco’s modified eagle medium (DMEM) supplemented with fetal bovine serum (FBS 5%) and penicillin/streptomycin (P/S). After three passages, the ASCs were harvested. Subsequently, flow cytometry and reverse transcriptase polymerase chain reaction (RT-PCR) were used to detect expression levels of NANOG and OCT4 to evaluate stemness. Then, a differentiation medium that included high-glucose DMEM supplemented with 10% FBS, dexamethasone (10 nM), sodium β-glycerophosphate (5 mM) and ascorbic acid (100 µM) was added. The cells were cultivated for four weeks, and the odontogenic medium was changed every two days. Cell differentiation was evaluated with Alizarin red staining and expressions of collagen I (COL1A1), dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP1). Results The ASCs were effectively and easily isolated. They were negative for CD45 and positive for the CD105 and CD73 markers. The ASCs expressed OCT4 and NANOG. Differentiated cells highly expressed DSPP, COL1A1 and DMP1. Alizarin red staining revealed a positive reaction for calcium deposition. ConclusionASCs were isolated successfully in high numbers from the buccal fat pad of human volunteers and were differentiated into odontoblast-like cells. These ASCs could be considered a new source of cells for use in regenerative endodontic treatments.

Establishing Mouse Models of B-Cell Non-Hodgkin's Lymphoma

There are numerous types of B-cell lymphoma including Burkitt lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma/mucosa-associated lymphoid tissue lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone lymphoma and other less frequent subtypes. However, the molecular pathogenesis of B-cell lymphoma remain largely unknown. Here we chose three different B-cell lymphoma subtype cell lines to establish mouse models, which could be useful to determine the efficacy and mechanisms of novel therapies.Firstly, we injected subcutaneously lymphoma cells into the right axillary region of BALB/c (nu/nu) nude mice. DOHH2 F0, Ly8 F0, Raji F0 appeared 7, 7, 21 days later. When tumors growed to 1.5cm at length, successful passage of mouse models was established by injecting cell suspension obtained from mincing and grinding tumors through a 70-μm cell strainer. The cell suspension was cultured at 37°C in a humidified atmosphere containing 5% CO2. The cells were cryopreserved in cryopreservation-medium (90% FBS, 10% DMSO), stored in liquid nitrogen, thawed again (with a viability of more than 70%) and successfully reconstituted. The above cell lines mouse models have been passaged to the 16th, 18th, 13th generation.For histological examination, an autopsy was performed on each passage of nude mice, and immunohistochmistry was use to determine the expression of surface markers. The tumor tissues from DOHH2 F0, DOHH2 F6, DOHH2 F12 were positive for CD20, cytoplasmic CD3, CD10, CD5, CD21, Ki67, CyclinD1, Bcl-2, Bcl-6. EBV was also detected in situ hybridization. Grade 3B Follicular lymphoma was identified in this B-cell lymphoma and was consistent with the primary origination of DOHH2. Immunohistochemical staining of Ly8 F0, Ly8 F6, Ly8 F12 showed the large atypical cells were positive for CD20, CD3, CD10, Bcl-2, mum-1, no reactivity to antibodies for bcl-6 and negative to EBER. Ki-67 values were graded as 30%. The pathologic expression in Raji F0, Raji F6, Raji F12 is like the following: CD20+/CD3+-/Ki67+-/CD10+/Bcl2+-/ Bcl6- /Mum1+/EBER++.Flow cytometry analysis was performed on cell suspensions cultured from each passage tumor tissues. DOHH2 F0, DOHH2 F6,DOHH2 F12 cells were CD19+/CD20-/CD10+/CD5-/CD38+/HLA-DR+/ CD22+ /CD23+/Ki-67+CD71+, and the kappa light chain was expressed monoclonally. Similar to DOHH2 cells, the serial Ly8 cells were CD19+ /CD20-/CD10+/CD5-/CD38+/HLA-DR+/CD22+/CD23+Ki-67+CD71+/EBER-, and the kappa light chain was expressed monoclonally. As Burkitt lymphoma, Raji and its serial cell clones were CD19+/CD20+/CD10+/CD5-/CD38+/HLA-DR+/CD22+/Ki-67+/CD71+/EBER++.In conclusions, we firstly and systematically established mouse models of B-cell lymphoma and take into passage successfully. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Molecular characteristics and tumorigenicity of ascites-derived tumor cells: mitochondrial oxidative phosphorylation as a novel therapy target in ovarian cancer.

Ovarian cancer disseminates primarily intraperitoneally. Detached tumor cell aggregates (spheroids) from the primary tumor are regarded as ‘metastatic units’ that exhibit a low sensitivity to classical chemotherapy, probably due to their unique molecular characteristics. We have analyzed the cellular composition of ascites from OvCa patients, using flow cytometry, and studied their behavior in vitro and in vivo. We conclude that ascites‐derived cultured cells from OvCa patients give rise to two subpopulations: adherent cells and non‐adherent cells. Here, we found that the AD population includes mainly CD90+ cells with highly proliferative rates in vitro but no tumorigenic potential in vivo, whereas the NAD population contains principally tumor cell spheroids (EpCAM+/CD24+) with low proliferative potential in vitro. Enriched tumor cell spheroids from the ascites of high‐grade serous OvCA patients, obtained using cell strainers, were highly tumorigenic in vivo and their metastatic spread pattern precisely resembled the tumor dissemination pattern found in the corresponding patients. Comparative transcriptome analyses from ascites‐derived tumor cell spheroids (n = 10) versus tumor samples from different metastatic sites (n = 30) revealed upregulation of genes involved in chemoresistance (TGM1, HSPAs, MT1s), cell adhesion and cell‐barrier integrity (PKP3, CLDNs, PPL), and the oxidative phosphorylation process. Mitochondrial markers (mass and membrane potential) showed a reduced mitochondrial function in tumoroids from tumor tissue compared with ascites‐derived tumor spheroids in flow cytometry analysis. Interestingly, response to OXPHOS inhibition by metformin and IACS010759 in tumor spheroids correlated with the extent of mitochondrial membrane potential measured by fluorescence‐activated cell sorting. Our data contribute to a better understanding of the biology of ovarian cancer spheroids and identify the OXPHOS pathway as new potential treatment option in advanced ovarian cancer.

Creating Matched In vivo/In vitro Patient-Derived Model Pairs of PDX and PDX-Derived Organoids for Cancer Pharmacology Research.

Patient-derived tumor xenografts (PDXs) are considered the most predictive preclinical models, largely believed to be driven by cancer stem cells (CSC) for conventional cancer drug evaluation. A large library of PDXs is reflective of the diversity of patient populations and thus enables population based preclinical trials ("Phase II-like mouse clinical trials"); however, PDX have practical limitations of low throughput, high costs and long duration. Tumor organoids, also being patient-derived CSC-driven models, can be considered as the in vitro equivalent of PDX, overcoming certain PDX limitations for dealing with large libraries of organoids or compounds. This study describes a method to create PDX-derived organoids (PDXO), thus resulting in paired models for in vitro and in vivo pharmacology research. Subcutaneously-transplanted PDX-CR2110 tumors were collected from tumor-bearing mice when the tumors reached 200-800 mm3, per an approved autopsy procedure, followed by removal of the adjacent non-tumor tissues and dissociation into small tumor fragments. The small tumor fragments were washed and passed through a 100 µm cell strainer to remove the debris. Cell clusters were collected and suspended in basement membrane extract (BME) solution and plated in a 6-well plate as a solid droplet with surrounding liquid media for growth in a CO2 incubator. Organoid growth was monitored twice weekly under light microscopy and recorded by photography, followed by liquid medium change 2 or 3 times a week. The grown organoids were further passaged (7 days later) at a 1:2 ratio by disrupting the BME embedded organoids using mechanical shearing, aided by addition of trypsin and the addition of 10 µM Y-27632. Organoids were cryopreserved in cryo-tubes for long-term storage, after release from BME by centrifugation, and also sampled (e.g., DNA, RNA and FFPE block) for further characterization.

Human Visceral Adipose Tissue (VAT) Inflammation Is Not Associated With Perforin Deficiency in Type 2 Diabetes Mellitus (DM) Subjects as Compared to Health Controls

Abstract Introduction: Role of T cells in VAT inflammation is poorly characterised. Perforin deficiency is associated with autoimmune inflammatory diseases like Hemophagocytosis Lymphohistiocytosis,as also in context of type 1 DM pathogenesis. Data from animal models suggest that perforin deficiency leads to VAT inflammation1Objective: We hypothesized CD8+/perf+ and CD56+/perf+ cells to be decreased in type 2 DM as compared to healthy controls. The present study also explored the difference in activation of T/NK cells between two groups Methods: 2×2 cm omental tissue was obtained from subjects undergoing elective abdominal surgery. The sample was transported in RPMI solution and stored in -80 ℃. Processing involved thawing, incubation at 37.6 ℃ for 24 hours with type IV Collagenase (1 mg/ml, Sigma Aldrich) 1ml/g of tissue,centrifuge (32g for 10 min at 10℃). The resultant Stromal Vascular fraction (SVF) was suspended in phosphate buffer saline (PBS), passed through cell strainer to make single cell suspension. It was again centrifuged and tagged with CD markers of interest. Fc block was added and single cell solution with FACS fluid prepared. It was run in BD CANTO-2 flow cytometer as described2Results: Of seventeen samples analysed, twelve samples of type 2 diabetes subjects were compared with five healthy controls. All results are presented in median. The diabetics had higher HbA1c (8.1 % vs 6%), higher BMI (28 kg/m2 vs 24 kg/m2), hsCRP(2.1 mg/dl Vs 0.9 mg/dl) but there was no difference in HOMA-Ir (5 vs 5.2 mU/L/mg/dl). The percentage of CD4+ + CD8+ cells/g of VAT was similar in both cases and control (20 × 103 Vs 23× 103). CD8+/CD45+/perf+ and CD56+/CD45+/perf+ could not be identified in any of the samples. Although CD8+/CD45+/perf-- and CD56+/CD45+/perf-- cells were identified. Next, we analysed the same cells for cytotoxic activation by 107a. The percentage of 107a positivity was low in CD 8+ (7% and 4 % respectively in cases and control) and CD 56+ cells (10% and 9 % respectively in cases and control),Although clinically type 2 DM subjects were obese and had inflammation (i.e higher hsCRP), there was no difference in VAT activation of immune cells studied. Also, we could not delineate perforin in any of the samples. Conclusion: Taken together this work suggests VAT T cell immune milieu in human Type 2 DM is different from mouse model. It is neither characterised by perforin deficiency nor activation of T cell/NK cell. This study points towards the probability that, the role of T cell/NK cells in human VAT infiltration could be fundamentally different from mice models. Further studies should be focussed on functional characteristics of these cells and interaction with VAT macrophages.

A modified protocol for isolation of retinal microglia from the pig

Microglia are the resident immune cells in the retina. To investigate their properties and behaviour, a reliable and yielding procedure to culture them is necessary. We here describe a way of isolation of microglial cells from the porcine retina, as pig eyes are similar to human eyes in size, structure and vasculature, including similarities in proteins and pathways. Retina was isolated from fresh pig eyes, dissociated by a mixture of collagenase, hyaluronidase and DNAse, and passed through a cell strainer. After triple centrifugation with decreasing velocity and re-suspension, cells were seeded into poly-d-lysine coated culture flasks and cultured using DMEM and macrophage-colony stimulating factor (M-CSF). Number of cells increased gradually during the first 10–14 days, till they could be split and used for experiments. Identity of isolated cells as microglia was assessed by immunostaining against the microglia/macrophage markers Iba1, CD11b, CD68, CD45 and TMEM119. Phagocytic function of microglia could be demonstrated by phagocytosis of fluorescence beads and their response to lipopolysaccharide (LPS). As a conclusion, we developed a protocol for isolation and cultivation of pig retinal microglial cells that are suitable for research in the laboratory.

Mesenchymal stromal cell treatment of donor kidneys during ex vivo normothermic machine perfusion: A porcine renal autotransplantation study.

Normothermic machine perfusion (NMP) of injured kidneys offers the opportunity for interventions to metabolically active organs prior to transplantation. Mesenchymal stromal cells (MSCs) can exert regenerative and anti-inflammatory effects in ischemia-reperfusion injury. The aims of this study were to evaluate the safety and feasibility of MSC treatment of kidneys during NMP using a porcine autotransplantation model, and examine potential MSC treatment-associated kidney improvements up to 14days posttransplant. After 75min of kidney warm ischemia, four experimental groups of n=7 underwent 14h of oxygenated hypothermic machine perfusion. In three groups this was followed by 240min of NMP with infusion of vehicle, 10million porcine, or 10million human adipose-derived MSCs. All kidneys were autotransplanted after contralateral nephrectomy. MSC treatment did not affect perfusion hemodynamics during NMP or cause adverse effects at reperfusion, with 100% animal survival. MSCs did not affect plasma creatinine, glomerular filtration rate, neutrophil gelatinase-associated lipocalin concentrations or kidney damage assessed by histology during the 14days, and MSCs retention was demonstrated in renal cortex. Infusing MSCs during ex vivo NMP of porcine kidneys was safe and feasible. Within the short posttransplant follow-up period, no beneficial effects of ex vivo MSC therapy could be demonstrated.

Amelanotic B16-F10 Melanoma Compatible with Advanced Three-Dimensional Imaging Modalities

Amelanotic B16-F10 Melanoma Compatible with Advanced Three-Dimensional Imaging Modalities

In Vitro Stem Cell Isolation from Human Breast Cancer

Background: Breast cancer (BC) is the most prevalent cancer among women worldwide with significant incidence and death rates. Nowadays, researchers hold that tumor formation, failure in therapy, and disease progression are all related to the presence of a small fraction of cancer cells with self-renewal capability known as “breast cancer stem cells” (BCSCs). Therefore, the study of this cancer cell population can be conducive to eradicating the tumor. The objective of the present study was to survey the existence and in vitro isolation of human BCSCs. Method: An in vitro research study was conducted under controlled laboratory settings to isolate, enrich, and identify breast cancer stem cells. Briefly, fresh breast tumors were carried to the lab immediately after surgery, followed by mechanical and enzymatic digestion (2 mg/ml collagenase I). Then, digested samples were passed through cell strainers (70 and 40 μm), and obtained cell suspension was cultured under the serum-free medium supplemented with growth factors for 21 days. The expression of cluster of differentiation 44 (CD44) and cluster of differentiation 24 (CD24) surface markers was assessed using immunocytochemistry, and stem cell gene expression was analyzed via RT-PCR. Results: BCSCs were able to survive in serum-free conditions and form floating spheres in vitro. Cells obtained from mammospheres expressed CD44 as the membranous and cytoplasmic pattern while CD24 expression was negative. Also, octameter-binding transcription factor 4 and SOX2 gene expression was observed in BCSCs. Conclusion: The presence of stem cells was confirmed in Iranian women BC, and an efficient in vitro mammosphere culture model was used to enrich and propagate BCSCs. In our opinion, this in vitro model could be a suitable method for isolating and enriching BCSCs.

ILC3-derived acetylcholine promotes protease-driven allergic lung pathology

ILC3-derived acetylcholine promotes protease-driven allergic lung pathology

CXCR4 expression by mesenchymal stromal cells is lost after use of enzymatic dissociation agents, but preserved by use of non-enzymatic methods.

In recent years, multipotent mesenchymal stromal cells (MSCs) have demonstrated tremendous potential for use in regenerative medicine. CXCR4, the receptor for CXCL12, is highly expressed by bone marrow (BM) MSCs and the CXCR4/CXCL12 axis has been shown to be important for migration and homing of BM-MSCs. Typically, MSCs used for clinical applications are collected after culture expansion using enzymatic methods, such as trypsin. Here, we compared different commercially available enzymatic and non-enzymatic methods for collection and dissociation of MSCs from culture plastics and their effects on CXCR4 expression by MSCs. We found that whereas non-enzymatic dissociation buffers and methods maintained CXCR4 expression, all tested enzymatic dissociation solutions dramatically decreased expression of CXCR4. We, therefore, strongly recommend the use of non-enzymatic dissociation methods, followed by filtration through a cell strainer to obtain single cell suspensions, in order to preserve maximal CXCR4 expression and optimal homing of cells.

Correction to: Preservation of connexin 43 and transzonal projections in isolated bovine pre-antral follicles before and following vitrification

Gap junctions and transzonal projections play a crucial role in intercellular communication between different follicular components and are necessary for follicle development. We aimed to demonstrate gap junction protein connexin 43 (Cx43) and transzonal projections (TZPs) in viable, category 1, isolated bovine pre-antral follicles (PAFs) during short-term culture and after vitrification and warming. This study involved four experimental groups: fresh control, 2-day culture, 4-day culture, and vitrified secondary PAFs. Isolated PAFs were vitrified using a simple and efficient cryopreservation method by means of mini cell strainers. Cx43 and TZPs were detected in pre-antral follicles of all stages, as well as in every experimental group. The group fresh follicles showed a higher percentage of follicles that were positive for Cx43 (91.7%) than the follicles that were vitrified (77.4%). All follicles that were cultured for 2 days were Cx43-positive (100%). Follicles cultured for 4 days (65.8%) (P = 0.002) showed the lowest percentage of follicles that were Cx43-positive. The percentages of the presence or (partial) absence of the TZP network were shown to be very heterogeneous between follicles in different treatment groups. These results suggest the maintenance of communication between the oocyte and the somatic companion cells after vitrification and warming. The varying percentages of the expression of the TZP network within groups suggests that it will be of interest to investigate whether this is truly due to variability in TZP integrity and follicle quality or due to methodological limitations.

Preservation of connexin 43 and transzonal projections in isolated bovine pre-antral follicles before and following vitrification.

Gap junctions and transzonal projections play a crucial role in intercellular communication between different follicular components and are necessary for follicle development. We aimed to demonstrate gap junction protein connexin 43 (Cx43) and transzonal projections (TZPs) in viable, category 1, isolated bovine pre-antral follicles (PAFs) during short-term culture and after vitrification and warming. This study involved four experimental groups: fresh control, 2-day culture, 4-day culture, and vitrified secondary PAFs. Isolated PAFs were vitrified using a simple and efficient cryopreservation method by means of mini cell strainers. Cx43 and TZPs were detected in pre-antral follicles of all stages, as well as in every experimental group. The group fresh follicles showed a higher percentage of follicles that were positive for Cx43 (91.7%) than the follicles that were vitrified (77.4%). All follicles that were cultured for 2days were Cx43-positive (100%). Follicles cultured for 4days (65.8%) (P = 0.002) showed the lowest percentage of follicles that were Cx43-positive. The percentages of the presence or (partial) absence of the TZP network were shown to be very heterogeneous between follicles in different treatment groups. These results suggest the maintenance of communication between the oocyte and the somatic companion cells after vitrification and warming. The varying percentages of the expression of the TZP network within groups suggests that it will be of interest to investigate whether this is truly due to variability in TZP integrity and follicle quality or due to methodological limitations.

The Quick Antigen Identification By Tissue Flow Cytometry for CAR-T Therapy in Relapsed/Refractory B-Cell Malignancies without Bone Marrow Involvement or Serous Cavity Effusions

In recent years, chimeric antigen receptor (CAR) T-cell therapy has been widely used to treat relapsed/refractory B-cell malignancies. Before CAR-T, the identification of target antigens on tumor cells is needed for choosing a suitable antigen-specific CAR, which is usually performed by multiparameter flow cytometry (FCM) for bone marrow (BM) samples or immunohistochemical (IHC) staining for tissue samples. However, some patients with tumor masses only and without BM involvement or serous cavity effusions (SCE) may not have much time to wait the results from IHC staining owing to rapidly growing tumor cells, therefore, we established a quick antigen identification by FCM using tissue samples. Fresh tumor tissues taken by fine or coarse needle puncture from different body parts were put in saline with 2% fetal calf serum and sent to the FCM lab as quickly as possible, once in lab, samples were processed immediately. Tissue strips were placed into a 60 mm petri dish, gently pressed by a syringe piston and repeatedly flushed with saline to separate cells from tissues; then the liquid with cells in petri dish were transferred into a 100μm cell strainer on top of 50 mL conical tube to exclude non-cellular tissue components, the strainer was washed with saline for 2-3 times. The strained cell suspension in conical tube was centrifuged at 1000rpm for 5 minutes, the supernatant was discarded by pipetting, cells were counted and resuspended with saline at a concentration of 104-105/ml, then the single cell suspension was ready for flow cytometry assay. 7-AAD and antibodies CD19/CD22/CD7/CD45 were added into the first tube to quantify viable cells and identify cells available for analysis, if dead cells were more than 30% of all cells, 7-AAD was needed for all next tubes, otherwise, it could be omitted. The standard antibody panels consist of CD10/CD20/CD34/CD19/CD38/CD45 for precursor B-cells and kappa/lambda/CD10/CD19/CD20/CD38/CD45 for mature B-cells, extra antibodies could be added according to patient's situations. Samples were performed on the 8-color BD FACSCanto II flow cytometer, and analyzed by BD FACS Diva software or Kaluza software from Beckman Coulter. Antigen expression, partial expression and no expression were defined as >80%, 20-80% and <20% of gated cells displaying interested antigen, respectively. Dim expression was defined as more than one log weaker than the normal counterpart's mean fluorescence intensity (MFI). From October 2017 through June 2020, a total of 63 patients with relapsed/refractory B-cell malignancies who had tumor masses only and no BM involvement or malignant SCE were hospitalized in our center for CAR-T treatment after failure of multiple-line therapies, including 11 (17.5%) acute lymphoblastic leukemia (ALL) and 52 (82.5%) non-Hodgkin's lymphoma (NHL), the median age was 41 years (range, 1-71) with both adults (n=47, 74.6%) and children younger than 18 years (n=16, 25.4%). Acquired events from tissue samples varied from 1000 to more than 100, 000 each tube (5 cases <5000 and 17 cases >100,000), the percentages of tumor cells in all cells available for analysis varied from 0.03% to 99.44% (5 cases <5% and 13 cases >90%). We have CD19-, CD20- and CD22-specific CAR-T cells for B-ALL and B-NHL, the choice of a CAR-T product depends on the antigen expression on tumor cells, patients with partial or no or dim expression (PND expression) of certain antigen are not suitable for the relevant antigen-specific CAR-T therapy. Among this cohort of patients, CD19 expressed in all but 1 ALL case who had accepted CD19 CAR-T; PND expression of CD20 occurred in 19.2% (10/52) of NHL patients since they were repeatedly administrated anti-CD20 antibodies in previous treatments which resulted in antigen diminishment or loss, all B-ALL patients had no or partial CD20 expression; PND expression of CD22 was seen in 18.2% (2/11) of ALL and 13.5% (7/52) of NHL. All flow results came back within hours (< 4-6 hours), much quicker than results from IHC staining which takes days. In conclusion, the correct selection of a target antigen is the basis of effective CAR-T treatment in relapsed/refractory B-cell malignancies especially for patients having accepted antibody or CAR-T therapies, our antigen detection by tissue flow cytometry provided a quick and accurate antigen identification for patients with no BM or SCE samples. Disclosures No relevant conflicts of interest to declare.

CXXC5 variant in an immunodeficient patient with a progressive loss of hematopoietic cells

Partial loss of function variant in CXXC5 identified in an immunodeficient patient with progressive bone marrow failure, corrected by bone marrow transplant.