human es cell line tc71  (DSMZ)

Journal: Frontiers in Cell and Developmental Biology

Article Title: A 3D Platform to Investigate Dynamic Cell-to-Cell Interactions Between Tumor Cells and Mesenchymal Progenitors

doi: 10.3389/fcell.2021.767253

Figure Lengend Snippet: ddPCR assays are able to simultaneously detect tumor cells and gene-modified MSCs. (A) , A 3-plex ddPCR assay was developed to quantitively evaluate tumor cells and MSCs in the VITVO50. Custom primers and probes were designed on Luc and GFP target genes to identify tumor cells and MSCs, respectively. hRPP30 was used as reference gene to estimate the total number of human cells. Due to the DNA distribution within the droplets, in the 3-plex ddPCR assay droplets were sorted out in eight different colored clusters. (B) and (C) , 4-plex ddPCR assays were set up to simultaneously investigate MSC biodistribution and antitumor effect in the ES metastatic model. gDNA was obtained from lungs and liver of mice. Because of the random distribution of DNA into the droplets, in 4-plex ddPCR assays droplets are clustered into sixteen different colored groups. The control 4-plex ddPCR assay (B) was developed combining Luc and GFP target assays with hRPP30 and mTfrc reference assays to detect human cells, TC71 Luc cells and control MSCs, localized in mice organs. The sTRAIL 4-plex ddPCR assay (C) was set up matching Luc and sTRAIL target assays with the mTfrc and hRPP30 reference assays to distinguish between TC71 Luc tumor cells and effector MSCs in mice organs.

Article Snippet: The human ES cell line TC71 (RRID: CVCL_2,213, DSMZ, Braunschweig, Germany) was cultivated in Iscove’s modified Dulbecco’s medium (IMDM, Euroclone, Milan, Italy) supplemented with 10% FBS (Carlo Erba Reagents Srl, Cornaredo, Italy), 1% L-glutamine (200 mM; BioWhittaker, Lonza, Verviers, Belgium), and 1% penicillin-streptomycin (104 UI/ml penicillin and 10 mg streptomycin/ml; Carlo Erba Reagents Srl).

Techniques: Modification, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: A 3D Platform to Investigate Dynamic Cell-to-Cell Interactions Between Tumor Cells and Mesenchymal Progenitors

doi: 10.3389/fcell.2021.767253

Figure Lengend Snippet: TC71 cell line and MSCs effectively express reporter genes. (A) , Gating strategy for the analysis of the GFP expression on MSCs transduced with GFP-encoding pCCL PGK WPRE lentiviral vector and sorted by FACS. Gate 1: forward scatter (FSC) and side scatter (SSC) area was used to enrich for intact cells (P1). Gate 2: GFP fluorescence recorded in the logarithmic scale was used to identify MSC GFP+ (P2; 93.9 ± 0.4%). (B) , Representative picture of GFP-expressing MSCs by fluorescence microscopy (original magnification ×100). (C) , Gating strategy for the analysis of the DsRed expression on TC71 Luc cells transduced with DsRed-encoding MIGR1 vector. Gate 1: FSC and SSC area was used to enrich for intact cells (P1). Gate 2: DsRed fluorescence assessed in the logarithmic scale allowed the identification of DsRed-expressing TC71 Luc cells (P2; 98.5 ± 0.7%). (D) , Representative picture of DsRed-expressing TC71 Luc cells by fluorescence microscopy (original magnification ×100).

Article Snippet: The human ES cell line TC71 (RRID: CVCL_2,213, DSMZ, Braunschweig, Germany) was cultivated in Iscove’s modified Dulbecco’s medium (IMDM, Euroclone, Milan, Italy) supplemented with 10% FBS (Carlo Erba Reagents Srl, Cornaredo, Italy), 1% L-glutamine (200 mM; BioWhittaker, Lonza, Verviers, Belgium), and 1% penicillin-streptomycin (104 UI/ml penicillin and 10 mg streptomycin/ml; Carlo Erba Reagents Srl).

Techniques: Expressing, Transduction, Plasmid Preparation, Fluorescence, Microscopy

Journal: Frontiers in Cell and Developmental Biology

Article Title: A 3D Platform to Investigate Dynamic Cell-to-Cell Interactions Between Tumor Cells and Mesenchymal Progenitors

doi: 10.3389/fcell.2021.767253

Figure Lengend Snippet: The VITVO50-based fluidic circuit allows to investigate dynamic cell-to-cell interactions between tumor nodules and MSCs. (A) , Scan of nine representative fields of the VITVO50 matrix colonized by TC71 Luc cells (red; Objective 4×). (B) , Representative picture taken at a higher magnification showing the formation of in vivo-like nodules of tumor cells in the VITVO50 matrix (red cell aggregates; Objective 10×) (C) Mean (SD) number of tumor cells colonizing VITVO50 bioreactor derived from escape data. Numbers of independent experiments n = 3. (D) , Bioluminescent signal after luciferin administration was used to quantify the viability of TC71 Luc cells seeded on the matrix. Data are expressed as the mean (SD). Numbers of independent experiments n = 3. (E) , Scan of nine representative fields of the VITVO50 matrix to evaluate interaction between TC71 Luc cells (red) and MSCs (green; Objective 4×). (F) , Representative picture taken at a higher magnification showing MSCs trapped between the fibers of VITVO50 matrix and interacting with tumor nodules (Objective 10×). (G) , Mean (SD) of the percentage of tumor cells (red) and MSCs (green) within the VITVO50 matrix obtained using the 3-plex ddPCR assay. Numbers of independent experiments n = 4. (H) , Ratio between the number of MSCs and tumor cells on VITVO50 matrix expressed as mean (SD). Numbers of independent experiments n = 4.

Article Snippet: The human ES cell line TC71 (RRID: CVCL_2,213, DSMZ, Braunschweig, Germany) was cultivated in Iscove’s modified Dulbecco’s medium (IMDM, Euroclone, Milan, Italy) supplemented with 10% FBS (Carlo Erba Reagents Srl, Cornaredo, Italy), 1% L-glutamine (200 mM; BioWhittaker, Lonza, Verviers, Belgium), and 1% penicillin-streptomycin (104 UI/ml penicillin and 10 mg streptomycin/ml; Carlo Erba Reagents Srl).

Techniques: In Vivo, Derivative Assay

Journal: Translational Oncology

Article Title: Anti-GD2 CAR MSCs against metastatic Ewing's sarcoma

doi: 10.1016/j.tranon.2021.101240

Figure Lengend Snippet: BF MSCs establish strong and stable connections with GD2-expressing ES cells. GD2 tCAR-mediated binding of MSCs to ES cell lines was investigated using cell-to-cell interaction assays. a-c , The number of MSC-ES cell aggregates, reported as the fold change versus EV MSCs, for all three ES cell lines. For TC71 * p < .001, ° p < .001; for A673 * p < .05, ° p < .001. d , The stability of the GD2 tCAR-mediated binding was examined by comparing the interactions of EV and GD2 tCAR MSCs with the TC71 ES line. MSC-TC71 aggregates were maintained at 4 °C on a rotating support for 2 and 4 h, and the number of aggregates was quantified by FACS. The number of MSC-TC71 aggregates at 2 and 4 h is reported as the fold change relative to the respective baseline number at the time of detachment (T0). * p < .05. All-p values were calculated by Student's t -test. Data are expressed as the mean (SD). Numbers of independent experiments n = 2, each with three technical replicates.

Article Snippet: The human ES cell lines TC71 (RRID: CVCL_2213, DSMZ, Braunschweig, Germany) and A673 (kind gift from Dr. Nicola Baldini, Rizzoli Orthopaedic Institute, Bologna, Italy) were cultivated in Iscove's modified Dulbecco's medium (IMDM, Euroclone, Milan, Italy).

Techniques: Expressing, Binding Assay

Journal: Translational Oncology

Article Title: Anti-GD2 CAR MSCs against metastatic Ewing's sarcoma

doi: 10.1016/j.tranon.2021.101240

Figure Lengend Snippet: BF MSCs and their conditioned supernatants exert in vitro cytotoxic effects against targeted ES cell lines. The in vitro impact of BF MSCs against the ES cell lines a , TC71 b , A673 and c , RD-ES was examined by co-cultures using multiple target:effector ratios (1:1; 1:2, and 1:5). Recombinant human TRAIL (rhTRAIL, 1 µg/ml) was used as a positive control, whereas tumour cells cultured alone were used as the negative control (CTR). Tumour cell death was examined by supravital propidium iodide (PI) after 24 h. Reported p-values represent the results of multiple comparisons between sTRAIL and BF MSC conditions and control groups, represented by EV MSCs, GD2 tCAR MSCs, rhTRAIL, or CTR. For TC71 * p < .001, ° p < .01, § p < .01; for A172 * p < .001, ° p < .001, § p < .001; for RD-ES * p < .001, ° p < .001, § p < .001. sTRAIL-mediated cytotoxicity against d , TC71 e , A673 and f , RD-ES cell lines. Tumour cells were incubated for 24 h with sTRAIL-containing supernatants (SNs) collected from sTRAIL and BF MSCs. SNs deriving from EV and GD2 tCAR MSCs were used as controls. ES cells in normal culture media (CTR) or treated with rhTRAIL (1 µg/ml) were evaluated for comparison. After 24 h, tumour cell death was assessed by supravital PI. For TC71 * p < .05, ° p < .001, § p < .05; for A673 * p < .001, ° p < .001, § p < .001; for RD-ES * p < .01, ° p < .001, § p < .001. All-p values have been calculated by Student's t -test. Data are expressed as the mean (SD). Numbers of independent experiments n = 2, each with three technical replicates.

Article Snippet: The human ES cell lines TC71 (RRID: CVCL_2213, DSMZ, Braunschweig, Germany) and A673 (kind gift from Dr. Nicola Baldini, Rizzoli Orthopaedic Institute, Bologna, Italy) were cultivated in Iscove's modified Dulbecco's medium (IMDM, Euroclone, Milan, Italy).

Techniques: In Vitro, Recombinant, Positive Control, Cell Culture, Negative Control, Control, Incubation, Comparison

Journal: Translational Oncology

Article Title: Anti-GD2 CAR MSCs against metastatic Ewing's sarcoma

doi: 10.1016/j.tranon.2021.101240

Figure Lengend Snippet: BF MSCs display potent antitumour activities against ES spheroid models. a , Tumour spheroids were established using DsRed-expressing TC71 and A673 cells (red) and treated with MSCs labelled with CFSE (green). Tumour spheroids were monitored for MSC infiltration and cytotoxicity for up to 48 h of co-culture by fluorescence microscopy and frozen sections at deep levels obtained by cryostat serial cutting. Representative images of the TC71 cell line are shown (magnification 50x, columns 1–3, and 100x, columns 4 and 5). The 15-hour time point was identified as the optimal time to quantify the antitumour effects of BF MSCs on TC71 and A673 spheroids, in terms of both reduced cell viability ( b and d ) and caspase-8 activation ( c and e ) by luminescence-based assays. For cell viability assay, TC71 * p < .001, ° p < .01, § p < .01; A673 * p < .001, ° p < .01, § p < .01. For caspase-8 activation assay TC71 * p < .01, ° p < .001, § p < .001; A673 * p < .01, ° p < .001, § p < .001. f and g , A 3D fibre-based matrix was employed to better model the tumour architecture. Bioluminescent signal after luciferin administration was used to quantify the viability of TC71 cells seeded on the matrix. The killing capacity of BF MSCs in co-culture with TC71 Luc (T:E ratio of 3:1) was assessed in a time-course experiment ( f , * p < .01, ° p < .05) and an endpoint assay after 4 days ( g , * p < .01, ° p < .01). The BF MSC cytotoxic effect was compared to those of rhTRAIL alone (1 µg/ml) and MSCs expressing sTRAIL only. EV MSCs, GD2 tCAR MSCs, and TC71 cells alone (CTR) were used as negative controls. The basal bioluminescence of the 3D matrix loaded by TC71 Luc cells was assessed immediately before MSC seeding or rhTRAIL treatment (T0). All p-values were calculated by Student's t -test. Data are expressed as the mean (SD). Numbers of independent experiments n = 2, each with three technical replicates.

Article Snippet: The human ES cell lines TC71 (RRID: CVCL_2213, DSMZ, Braunschweig, Germany) and A673 (kind gift from Dr. Nicola Baldini, Rizzoli Orthopaedic Institute, Bologna, Italy) were cultivated in Iscove's modified Dulbecco's medium (IMDM, Euroclone, Milan, Italy).

Techniques: Expressing, Co-Culture Assay, Fluorescence, Microscopy, Activation Assay, Viability Assay, End Point Assay

Journal: Translational Oncology

Article Title: Anti-GD2 CAR MSCs against metastatic Ewing's sarcoma

doi: 10.1016/j.tranon.2021.101240

Figure Lengend Snippet: Establishment of an in vivo model that closely mimics metastatic ES. Two million TC71 Luc cells were intravenously injected into NSG mice. TC71 Luc cell engraftment was assessed by monitoring in vivo bioluminescence using the IVIS system. a , Ten minutes after TC71 Luc cell injection, the luminescence signal accumulated in the lung. b , Over the next few hours, the bioluminescence gradually disappeared as the cells dispersed and re-emerged 10 days later at various locations where tumours developed. The most common engraftment sites were the lungs, liver, and femur. c , At sacrifice, on day 13, metastases were quantified in extracted organs by IVIS. d and e , Tumour metastases (black arrows) were confirmed by H&E staining on lung ( d ) and liver ( e ) sections (magnification 100x).

Article Snippet: The human ES cell lines TC71 (RRID: CVCL_2213, DSMZ, Braunschweig, Germany) and A673 (kind gift from Dr. Nicola Baldini, Rizzoli Orthopaedic Institute, Bologna, Italy) were cultivated in Iscove's modified Dulbecco's medium (IMDM, Euroclone, Milan, Italy).

Techniques: In Vivo, Injection, Staining

Journal: Translational Oncology

Article Title: Anti-GD2 CAR MSCs against metastatic Ewing's sarcoma

doi: 10.1016/j.tranon.2021.101240

Figure Lengend Snippet: BF MSCs are able to reduce lung metastases in the ES metastatic model. The BF MSC antitumour potential was examined in a metastatic model of ES. a , Representative therapeutic schedule: TC71 Luc cells (two million) were intravenously (i.v.) injected into NSG mice ( n = 62). Starting four days after TC71 Luc cell injection, the animals were randomly divided into five groups for treatment: control group ( n = 22) received no treatment (CTR); the EV MSC group ( n = 10), GD2 tCAR MSC group ( n = 10), sTRAIL MSC group ( n = 10), and BF MSC group ( n = 10) received multiple ( n = 3) i.v. injections of one million of the respective gene-modified MSCs, which were administered every three days. Before the infusion, the third dose of gene-modified MSCs was labelled by DiR dye (8 µM; in red). After 13 days, the animals were sacrificed. Lungs and liver were extracted, maintained on dry ice, and stored at −80 °C. 4-plex ddPCR assays were performed on organ-derived gDNA to simultaneously detect the presence of various cell types. b and c , Ratios of TC71 Luc cells per µl to the total number of cells per µl were calculated for lungs and liver. For each mouse group, the median (interquartile range; IQR) values were derived and multiplied by 1000, and groups were compared in terms of metastatic growth in lungs ( b ) and liver ( c ). For the lungs, sTRAIL MSCs vs CTR, EV MSCs or GD2 tCAR MSCs * p < .05, BF MSCs vs CTR, EV MSCs or GD2 tCAR MSCs ° p < .05. All p-values were calculated by the Wilcoxon–Mann–Whitney test.

Article Snippet: The human ES cell lines TC71 (RRID: CVCL_2213, DSMZ, Braunschweig, Germany) and A673 (kind gift from Dr. Nicola Baldini, Rizzoli Orthopaedic Institute, Bologna, Italy) were cultivated in Iscove's modified Dulbecco's medium (IMDM, Euroclone, Milan, Italy).

Techniques: Injection, Control, Modification, Derivative Assay, MANN-WHITNEY