Cunha B et al. (NOV 2015)
Journal of biotechnology 213 97--108
Exploring continuous and integrated strategies for the up- and downstream processing of human mesenchymal stem cells.
The integration of up- and downstream unit operations can result in the elimination of hold steps,thus decreasing the footprint,and ultimately can create robust closed system operations. This type of design is desirable for the bioprocess of human mesenchymal stem cells (hMSC),where high numbers of pure cells,at low volumes,need to be delivered for therapy applications. This study reports a proof of concept of the integration of a continuous perfusion culture in bioreactors with a tangential flow filtration (TFF) system for the concentration and washing of hMSC. Moreover,we have also explored a continuous alternative for concentrating hMSC. Results show that expanding cells in a continuous perfusion operation mode provided a higher expansion ratio,and led to a shift in cells' metabolism. TFF operated either in continuous or discontinuous allowed to concentrate cells,with high cell recovery (>80%) and viability (>95%); furthermore,continuous TFF permitted to operate longer with higher cell concentrations. Continuous diafiltration led to higher protein clearance (98%) with lower cell death,when comparing to discontinuous diafiltration. Overall,an integrated process allowed for a shorter process time,recovering 70% of viable hMSC (>95%),with no changes in terms of morphology,immunophenotype,proliferation capacity and multipotent differentiation potential.
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产品类型：
产品号#：
70022
70071
产品名：
Poulsen C et al. (AUG 2015)
Toxicology letters 237 1 21--9
Differential cytotoxicity of long-chain bases for human oral gingival epithelial keratinocytes, oral fibroblasts, and dendritic cells.
Long-chain bases are present in the oral cavity. Previously we determined that sphingosine,dihydrosphingosine,and phytosphingosine have potent antimicrobial activity against oral pathogens. Here,we determined the cytotoxicities of long-chain bases for oral cells,an important step in considering their potential as antimicrobial agents for oral infections. This information would clearly help in establishing prophylactic or therapeutic doses. To assess this,human oral gingival epithelial (GE) keratinocytes,oral gingival fibroblasts (GF),and dendritic cells (DC) were exposed to 10.0-640.0 μM long-chain bases and glycerol monolaurate (GML). The effects of long-chain bases on cell metabolism (conversion of resazurin to resorufin),membrane permeability (uptake of propidium iodide or SYTOX-Green),release of cellular contents (LDH),and cell morphology (confocal microscopy) were all determined. GE keratinocytes were more resistant to long-chain bases as compared to GF and DC,which were more susceptible. For DC,0.2-10.0 μM long-chain bases and GML were not cytotoxic; 40.0-80.0 μM long-chain bases,but not GML,were cytotoxic; and 80.0 μM long-chain bases induced cellular damage and death in less than 20 min. The LD50 of long-chain bases for GE keratinocytes,GF,and DC were considerably higher than their minimal inhibitory concentrations for oral pathogens,a finding important to pursuing their future potential in treating periodontal and oral infections.
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产品类型：
产品号#：
70041
产品名：
Shinkuma S et al. (MAY 2016)
Proceedings of the National Academy of Sciences of the United States of America 113 20 5676--5681
Site-specific genome editing for correction of induced pluripotent stem cells derived from dominant dystrophic epidermolysis bullosa.
Genome editing with engineered site-specific endonucleases involves nonhomologous end-joining,leading to reading frame disruption. The approach is applicable to dominant negative disorders,which can be treated simply by knocking out the mutant allele,while leaving the normal allele intact. We applied this strategy to dominant dystrophic epidermolysis bullosa (DDEB),which is caused by a dominant negative mutation in the COL7A1 gene encoding type VII collagen (COL7). We performed genome editing with TALENs and CRISPR/Cas9 targeting the mutation,c.80688084delinsGA. We then cotransfected Cas9 and guide RNA expression vectors expressed with GFP and DsRed,respectively,into induced pluripotent stem cells (iPSCs) generated from DDEB fibroblasts. After sorting,90% of the iPSCs were edited,and we selected four gene-edited iPSC lines for further study. These iPSCs were differentiated into keratinocytes and fibroblasts secreting COL7. RT-PCR and Western blot analyses revealed gene-edited COL7 with frameshift mutations degraded at the protein level. In addition,we confirmed that the gene-edited truncated COL7 could neither associate with normal COL7 nor undergo triple helix formation. Our data establish the feasibility of mutation site-specific genome editing in dominant negative disorders.
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产品类型：
产品号#：
05850
05857
05870
05875
85850
85857
85870
85875
产品名：
mTeSR™1
mTeSR™1
N. Camviel et al. (nov 2022)
Journal for immunotherapy of cancer 10 11
Both APRIL and antibody-fragment-based CAR T cells for myeloma induce BCMA downmodulation by trogocytosis and internalization.
BACKGROUND Chimeric antigen receptor (CAR) T cell therapy targeting B cell maturation antigen (BCMA) on multiple myeloma (MM) produces fast but not long-lasting responses. Reasons for treatment failure are poorly understood. CARs simultaneously targeting two antigens may represent an alternative. Here,we (1) designed and characterized novel A proliferation inducing ligand (APRIL) based dual-antigen targeting CARs,and (2) investigated mechanisms of resistance to CAR T cells with three different BCMA-binding moieties (APRIL,single-chain-variable-fragment,heavy-chain-only). METHODS Three new APRIL-CARs were designed and characterized. Human APRIL-CAR T cells were evaluated for their cytotoxic function in vitro and in vivo,for their polyfunctionality,immune synapse formation,memory,exhaustion phenotype and tonic signaling activity. To investigate resistance mechanisms,we analyzed BCMA levels and cellular localization and quantified CAR T cell-target cell interactions by live microscopy. Impact on pathway activation and tumor cell proliferation was assessed in vitro and in vivo. RESULTS APRIL-CAR T cells in a trimeric ligand binding conformation conferred fast but not sustained antitumor responses in vivo in mouse xenograft models. In vitro trimer-BB$\zeta$ CAR T cells were more polyfunctional and formed stronger immune synapses than monomer-BB$\zeta$ CAR T cells. After CAR T cell-myeloma cell contact,BCMA was rapidly downmodulated on target cells with all evaluated binding moieties. CAR T cells acquired BCMA by trogocytosis,and BCMA on MM cells was rapidly internalized. Since BCMA can be re-expressed during progression and persisting CAR T cells may not protect patients from relapse,we investigated whether non-functional CAR T cells play a role in tumor progression. While CAR T cell-MM cell interactions activated BCMA pathway,we did not find enhanced tumor growth in vitro or in vivo. CONCLUSION Antitumor responses with APRIL-CAR T cells were fast but not sustained. Rapid BCMA downmodulation occurred independently of whether an APRIL or antibody-based binding moiety was used. BCMA internalization mostly contributed to this effect,but trogocytosis by CAR T cells was also observed. Our study sheds light on the mechanisms underlying CAR T cell failure in MM when targeting BCMA and can inform the development of improved treatment strategies.
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产品类型：
产品号#：
07801
17849
产品名：
EasySep™人CD271正选试剂盒 II
Foley JF et al. (APR 2005)
Journal of immunology (Baltimore,Md. : 1950) 174 8 4892--900
Roles for CXC chemokine ligands 10 and 11 in recruiting CD4+ T cells to HIV-1-infected monocyte-derived macrophages, dendritic cells, and lymph nodes.
We investigated roles for chemoattractants in dissemination of HIV-1 by examining the induction of T cell-active chemokines in HIV-1-infected human monocyte-derived macrophages and dendritic cells. Of the 12 chemokines analyzed,mRNAs for two,CXCL10 and CXCL11,ligands for the chemokine receptor CXCR3,were up-regulated in both cell types upon infection by HIV-1. Induction of these chemokine genes in infected cultures was dependent on both viral entry and reverse transcriptase activity,but not on the HIV-1 envelope glycoprotein. Conditioned medium from infected cells was chemotactic for freshly isolated human CD4+ T cells,and chemotaxis was abolished by pretreatment with an Ab against CXCR3. A lymph node from an HIV-1-infected individual expressed CXCL10 and CXCL11 mRNAs in the paracortex,including venules,as detected by in situ hybridization,whereas neither mRNA was detected after highly active antiretroviral therapy. Because CCR5 on CD4+ T cells is found predominantly on cells that also express CXCR3,these data implicate CXCL10 and CXCL11 in the recruitment of susceptible T cells to HIV-1-infected lymph nodes,macrophages,and dendritic cells. This recruitment might enhance the sequestration of T cells in infected lymphoid organs and the spread of infection between cells,contributing to the immunopathology of AIDS.
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产品类型：
产品号#：
15022
15062
产品名：
RosetteSep™人CD4+ T细胞富集抗体混合物
RosetteSep™人CD4+ T细胞富集抗体混合物
Kennah E et al. (MAY 2009)
Blood 113 19 4646--55
Identification of tyrosine kinase, HCK, and tumor suppressor, BIN1, as potential mediators of AHI-1 oncogene in primary and transformed CTCL cells.
AHI-1 is an oncogene often targeted by provirus insertional mutagenesis in murine leukemias and lymphomas. Aberrant expression of human AHI-1 occurs in cutaneous T-cell lymphoma (CTCL) cells and in CD4(+)CD7(-) Sezary cells from patients with Sezary syndrome. Stable knockdown of AHI-1 using retroviral-mediated RNA interference in CTCL cells inhibits their transforming activity in vitro and in vivo. To identify genes involved in AHI-1-mediated transformation,microarray analysis was performed to identify differentially expressed genes in AHI-1-suppressed CTCL cells. Fifteen up-regulated and 6 down-regulated genes were identified and confirmed by quantitative reverse transcription-polymerase chain reaction. Seven were further confirmed in a microarray analysis of CD4(+)CD7(-) Sezary cells from Sezary syndrome patients. HCK and BIN1 emerged as new candidate cooperative genes,with differential protein expression,which correlates with observed transcript changes. Interestingly,changes in HCK phosphorylation and biologic response to its inhibitor,dasatinib,were observed in AHI-1-suppressed or -overexpressed cells. The tumor suppressor BIN1 physically interacts with MYC in CTCL cells,which also exhibit differential MYC protein expression. In addition,aberrant expression of alternative splicing forms of BIN1 was observed in primary and transformed CTCL cells. These findings indicate that HCK and BIN1 may play critical roles in AHI-1-mediated leukemic transformation of human CTCL cells.
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产品类型：
产品号#：
15021
15061
产品名：
RosetteSep™人T细胞富集抗体混合物
RosetteSep™人T细胞富集抗体混合物
Cai S et al. (APR 2011)
Clinical cancer research : an official journal of the American Association for Cancer Research 17 8 2195--206
Humanized bone marrow mouse model as a preclinical tool to assess therapy-mediated hematotoxicity.
PURPOSE: Preclinical in vivo studies can help guide the selection of agents and regimens for clinical testing. However,one of the challenges in screening anticancer therapies is the assessment of off-target human toxicity. There is a need for in vivo models that can simulate efficacy and toxicities of promising therapeutic regimens. For example,hematopoietic cells of human origin are particularly sensitive to a variety of chemotherapeutic regimens,but in vivo models to assess potential toxicities have not been developed. In this study,a xenograft model containing humanized bone marrow is utilized as an in vivo assay to monitor hematotoxicity. EXPERIMENTAL DESIGN: A proof-of-concept,temozolomide-based regimen was developed that inhibits tumor xenograft growth. This regimen was selected for testing because it has been previously shown to cause myelosuppression in mice and humans. The dose-intensive regimen was administered to NOD.Cg-Prkdc(scid)IL2rg(tm1Wjl)/Sz (NOD/SCID/γchain(null)),reconstituted with human hematopoietic cells,and the impact of treatment on human hematopoiesis was evaluated. RESULTS: The dose-intensive regimen resulted in significant decreases in growth of human glioblastoma xenografts. When this regimen was administered to mice containing humanized bone marrow,flow cytometric analyses indicated that the human bone marrow cells were significantly more sensitive to treatment than the murine bone marrow cells and that the regimen was highly toxic to human-derived hematopoietic cells of all lineages (progenitor,lymphoid,and myeloid). CONCLUSIONS: The humanized bone marrow xenograft model described has the potential to be used as a platform for monitoring the impact of anticancer therapies on human hematopoiesis and could lead to subsequent refinement of therapies prior to clinical evaluation.
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产品类型：
产品号#：
03434
03444
04434
04444
84434
84444
产品名：
MethoCult™GF M3434
MethoCult™GF M3434
MethoCult™H4434经典
MethoCult™H4434经典
Garnache-Ottou F et al. (FEB 2005)
Blood 105 3 1256--64
Expression of the myeloid-associated marker CD33 is not an exclusive factor for leukemic plasmacytoid dendritic cells.
A new entity of acute leukemia coexpressing CD4(+)CD56(+) markers without any other lineage-specific markers has been identified recently as arising from lymphoid-related plasmacytoid dendritic cells (pDCs). In our laboratory,cells from a patient with such CD4(+)CD56(+) lineage-negative leukemia were unexpectedly found to also express the myeloid marker CD33. To confirm the diagnosis of pDC leukemia despite the CD33 expression,we demonstrated that the leukemic cells indeed exhibited pDC phenotypic and functional properties. In 7 of 8 other patients with CD4(+)CD56(+) pDC malignancies,we were able to confirm that the tumor cells expressed CD33 although with variable expression levels. CD33 expression was shown by flow cytometry,reverse transcriptase-polymerase chain reaction,and immunoblot analysis. Furthermore,CD33 monoclonal antibody stimulation of purified CD4(+)CD56(+) leukemic cells led to cytokine secretion,thus confirming the presence of a functional CD33 on these leukemic cells. Moreover,we found that circulating pDCs in healthy individuals also weakly express CD33. Overall,our results demonstrate that the expression of CD33 on CD4(+)CD56(+) lineage-negative cells should not exclude the diagnosis of pDC leukemia and underline that pDC-specific markers should be used at diagnosis for CD4(+)CD56(+) malignancies.
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产品类型：
产品号#：
15028
15068
产品名：
RosetteSep™ 人单核细胞富集抗体混合物
RosetteSep™人单核细胞富集抗体混合物
Weng Z et al. (JUL 2014)
Stem cells and development 23 14 1704--1716
A simple, cost-effective but highly efficient system for deriving ventricular cardiomyocytes from human pluripotent stem cells.
Self-renewable human pluripotent stem cells (hPSCs) serve as a potential unlimited ex vivo source of human cardiomyocytes (CMs) for cell-based disease modeling and therapies. Although recent advances in directed differentiation protocols have enabled more efficient derivation of hPSC-derived CMs with an efficiency of ∼50%-80% CMs and a final yield of ∼1-20 CMs per starting undifferentiated hPSC,these protocols are often not readily transferrable across lines without first optimizing multiple parameters. Further,the resultant populations are undefined for chamber specificity or heterogeneous containing mixtures of atrial,ventricular (V),and pacemaker derivatives. Here we report a highly cost-effective and reproducibly efficient system for deriving hPSC-ventricular cardiomyocytes (VCMs) from all five human embryonic stem cell (HES2,H7,and H9) and human induced PSC (hiPSC) (reprogrammed from human adult peripheral blood CD34(+) cells using nonintegrating episomal vectors) lines tested. Cardiogenic embryoid bodies could be formed by the sequential addition of BMP4,Rho kinase inhibitor,activin-A,and IWR-1. Spontaneously contracting clusters appeared as early as day 8. At day 16,up to 95% of cells were cTnT(+). Of which,93%,94%,100%,92%,and 92% of cardiac derivatives from HES2,H7,H9,and two iPSC lines,respectively,were VCMs as gauged by signature ventricular action potential and ionic currents (INa(+)/ICa,L(+)/IKr(+)/IKATP(+)); Ca(2+) transients showed positive chronotropic responses to $\$-adrenergic stimulation. Our simple,cost-effective protocol required the least amounts of reagents and time compared with others. While the purity and percentage of PSC-VCMs were comparable to a recently published protocol,the present yield and efficiency with a final output of up to 70 hPSC-VCMs per hPSC was up to 5-fold higher and without the need of performing line-specific optimization. These differences were discussed. The results may lead to mass production of hPSC-VCMs in bioreactors.
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产品类型：
产品号#：
02690
05850
05857
05870
05875
07913
09850
85850
85857
85870
85875
产品名：
StemSpan™CC100
Dispase（5 U/mL）
mTeSR™1
mTeSR™1
Ortega V et al. (MAR 2016)
Cancer genetics 209 3 82--6
Optimal strategy for obtaining routine chromosome analysis by using negative fractions of CD138 enriched plasma cells.
Fluorescence in situ hybridization (FISH) is superior to routine chromosome analysis (RCA) in detecting important prognostic genetic abnormalities in plasma cell dyscrasia (PCD); however,its sensitivity is hampered due to paucity of plasma cells (PC) in whole bone marrow (BM). Studies showed that the abnormality detection rate in enriched plasma cells (EPC) is greater than unselected plasma cells (UPC),but purification techniques are limiting to only FISH when sample volumes are inadequate. Not performing RCA may compromise patient care since RCA is equally important for detecting non-PC related abnormalities when the diagnosis is undefined. To resolve this critical issue,we designed a study where an immuno-magnetic CD138 enriched positive selection was used for FISH while the negative fraction (NF) was used to retrieve other myeloid elements for RCA. Parallel FISH studies were performed using UPC and CD138 EPC,while karyotyping was achieved using whole BM and discarded myeloid elements from the NF. Results showed that the abnormality rate of EPC was doubled compared to UPC for FISH,and CA displayed 100% success rate using the NF. PCD related chromosome abnormalities were confined to whole BM while non-PCD related abnormalities were found in both whole BM and NF. Our results demonstrate the feasibility of using the NF for RCA.
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产品类型：
产品号#：
21000
20119
20155
18387
18387RF
产品名：
RoboSep™- S
RoboSep™ 吸头组件抛光剂
RoboSep™分选试管套装（9个塑料管+吸头保护器）
Haraguchi Y et al. (DEC 2015)
Journal of Tissue Engineering and Regenerative Medicine 9 12 1363--1375
Simple suspension culture system of human iPS cells maintaining their pluripotency for cardiac cell sheet engineering.
In this study,a simple three-dimensional (3D) suspension culture method for the expansion and cardiac differentiation of human induced pluripotent stem cells (hiPSCs) is reported. The culture methods were easily adapted from two-dimensional (2D) to 3D culture without any additional manipulations. When hiPSCs were directly applied to 3D culture from 2D in a single-cell suspension,only a few aggregated cells were observed. However,after 3 days,culture of the small hiPSC aggregates in a spinner flask at the optimal agitation rate created aggregates which were capable of cell passages from the single-cell suspension. Cell numbers increased to approximately 10-fold after 12 days of culture. The undifferentiated state of expanded hiPSCs was confirmed by flow cytometry,immunocytochemistry and quantitative RT-PCR,and the hiPSCs differentiated into three germ layers. When the hiPSCs were subsequently cultured in a flask using cardiac differentiation medium,expression of cardiac cell-specific genes and beating cardiomyocytes were observed. Furthermore,the culture of hiPSCs on Matrigel-coated dishes with serum-free medium containing activin A,BMP4 and FGF-2 enabled it to generate robust spontaneous beating cardiomyocytes and these cells expressed several cardiac cell-related genes,including HCN4,MLC-2a and MLC-2v. This suggests that the expanded hiPSCs might maintain the potential to differentiate into several types of cardiomyocytes,including pacemakers. Moreover,when cardiac cell sheets were fabricated using differentiated cardiomyocytes,they beat spontaneously and synchronously,indicating electrically communicative tissue. This simple culture system might enable the generation of sufficient amounts of beating cardiomyocytes for use in cardiac regenerative medicine and tissue engineering.
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EasySep™小鼠TIL（CD45）正选试剂盒
科学海报A Novel Animal Component-Free Culture System for Isolation and Expansion of Human Bone Marrow-Derived Mesenchymal Cells
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