Human umbilical vein endothelial cells (HUVEC) (C2519A; Lonza) and human mesenchymal stem cells (PT-2501; Lonza PT-2501) were grown as per the manufacture’s instruction. For reaggregation experiments a total of 1,000 sBC were sorted and reaggregated with 100 human mesenchymal stem cells and 400 HUVEC for 2 days in round bottom plates in a 50:50 mixture of maturation and HUVEC culture media as previously described (35 (link)).
Pt 2501
Manufactured by Lonza
Sourced in United States, Switzerland
The PT-2501 is a laboratory equipment designed for general scientific applications. It serves as a precision measurement tool, capable of performing various tasks as required by the user's needs. The core function of the PT-2501 is to provide accurate and reliable data collection and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Pt 3001, by Lonza (11 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Pt 3238, by Lonza (5 mentions)
Tryple express, by Thermo Fisher Scientific (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (5 mentions)
β glycerophosphate, by Merck Group (4 mentions)
Fetal bovine serum (fbs), by American Type Culture Collection (3 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Glutamax, by Thermo Fisher Scientific (3 mentions)
Hmscs, by Lonza (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Cc 2538, by Lonza (3 mentions)
Dexamethasone (dex), by Merck Group (3 mentions)
Pt 4106e, by Lonza (2 mentions)
Pt 4107e, by Lonza (2 mentions)
Mscgm bulletkit medium, by Lonza (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Msc growth medium, by Lonza (2 mentions)
78 protocols using pt 2501
1
Reaggregation of HUVEC, MSC, and sBC
2
Murine CD4+ T Cell Proliferation Assay
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A cell suspension of splenocytes from 6- to 8-week-old male mice was prepared, and the
erythrocytes were lysed with ACK (R7757, Sigma-Aldrich). The CD4+ T cells were
purified by using a CD4+ T-cell Isolation kit (130-104-454, Miltenyi Biotech)
according to the manufacturer’s instructions. The purity of the CD4+ T cells
was confirmed using a CD4 antibody (FITC-conjugated anti-mouse CD4, 553729, BD
Biosciences) and was greater than 96%. For the T-cell proliferation assay, CD4+T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; 565082,
BD Biosciences). Freshly purified T cells were labeled with 1.2 μM CFSE for 10 min at 37°C
and were then washed three times in complete medium. CFSE-labeled CD4+ T cells
were co-cultured with adult SCs, ESC-derived SLCs, human bone marrow-derived mesenchymal
stem cells (BM-MSC, PT-2501, Lonza, Allendale, NJ), or isolated spleen cells at a 5:1
ratio. The cells were co-cultured at 37°C for 5 days in RPMI1640 supplemented with 10% FBS
(all from Life Technologies), 100 units/ml penicillin and 100 μg/ml streptomycin
(Hyclone), 50ng/ml phorbol myristate acetate (PMA, P8139, Sigma-Aldrich), and 1μM
ionomycin (P9657, Sigma-Aldrich). After co-culture, the proliferation of the
CD4+ T cells was analyzed by assessing the intensity of the CFSE signal as
determined via flow cytometry (Calibur, BD Biosciences).
erythrocytes were lysed with ACK (R7757, Sigma-Aldrich). The CD4+ T cells were
purified by using a CD4+ T-cell Isolation kit (130-104-454, Miltenyi Biotech)
according to the manufacturer’s instructions. The purity of the CD4+ T cells
was confirmed using a CD4 antibody (FITC-conjugated anti-mouse CD4, 553729, BD
Biosciences) and was greater than 96%. For the T-cell proliferation assay, CD4+T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; 565082,
BD Biosciences). Freshly purified T cells were labeled with 1.2 μM CFSE for 10 min at 37°C
and were then washed three times in complete medium. CFSE-labeled CD4+ T cells
were co-cultured with adult SCs, ESC-derived SLCs, human bone marrow-derived mesenchymal
stem cells (BM-MSC, PT-2501, Lonza, Allendale, NJ), or isolated spleen cells at a 5:1
ratio. The cells were co-cultured at 37°C for 5 days in RPMI1640 supplemented with 10% FBS
(all from Life Technologies), 100 units/ml penicillin and 100 μg/ml streptomycin
(Hyclone), 50ng/ml phorbol myristate acetate (PMA, P8139, Sigma-Aldrich), and 1μM
ionomycin (P9657, Sigma-Aldrich). After co-culture, the proliferation of the
CD4+ T cells was analyzed by assessing the intensity of the CFSE signal as
determined via flow cytometry (Calibur, BD Biosciences).
3
Culture and Expansion of Human Mesenchymal Stem Cells
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Human mesenchymal stem cells (hMSCs, PT-2501, Lonza) were obtained from randomly selected healthy donors of both sexes with ages ranging from 19 to 38 years old and cultured in dedicated medium MSCBM (PT-3238) supplemented with 10% MCGS (PT-4106E), L-glutamine (PT-4107E), and gentamicin sulfate (GA-1000, PT-4504E, all Lonza), which was changed twice a week. Cells were maintained for up to 9 passages in 75 cm2 plastic bottom flasks and split once a week. For transgene induction experiments, cells were transferred to 24-well plates and seeded at a density of 1.5 × 104 cells/well. HEK293 cells served as controls and were cultivated in DMEM medium (31966 GlutaMAX, GIBCO) with 10% FBS (P40-37500HI, PAN™Biotech) and penicillin/streptomycin (P11-010, PAA). Cells were grown in 25 cm2 flasks, but for transfections assays were transferred to 24-well plates at a density of 70 × 103 cells/well. Both cell types were cultured in a humidified atmosphere at 37 °C and 5% CO2.
4
Isolation of Human MSC-Derived Extracellular Vesicles
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Human mesenchymal stem cells (MSC) were purchased from Lonza (Walkersville, MD, USA, #PT-2501) and cultured in a 175 cm2 flask with 25 mL mesenchymal stem cell media (Lonza, Walkersville, MD, USA, #PT-3001) in a humidified incubator at 37 °C with 5% CO2, according to manufacturer’s protocol. The cell propagation was limited to passage 10.
After reaching 80–90% confluency (about 1.5–2 million cells/flask), human MSCs were washed with 15mL PBS followed by adding 25 mL serum-free RPMI (Life Technologies) medium for another 24 h. MSC-derived EVs were collected from the culture medium by differential centrifugation at 2000 × g for 30 min to remove cell debris, and then, 100,000 × g for 1 h, collecting of the 100,000 × g pellet after. EVs were stored in PBS supplemented with 1% DMSO at −80 °C until EV DiD dye labeling. We used WX Ultra Centrifuge with Sorvall AH-629 rotor.
After reaching 80–90% confluency (about 1.5–2 million cells/flask), human MSCs were washed with 15mL PBS followed by adding 25 mL serum-free RPMI (Life Technologies) medium for another 24 h. MSC-derived EVs were collected from the culture medium by differential centrifugation at 2000 × g for 30 min to remove cell debris, and then, 100,000 × g for 1 h, collecting of the 100,000 × g pellet after. EVs were stored in PBS supplemented with 1% DMSO at −80 °C until EV DiD dye labeling. We used WX Ultra Centrifuge with Sorvall AH-629 rotor.
5
Comparative Analysis of hMSC Cell Lines
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Two hMSC cell lines obtained from the bone marrow of healthy donors, provided by two different commercial suppliers – PT-2501 from a 21-year-old female and a 22-year-old female (Lonza Group Ltd, Basel, Switzerland), and C-12974 from a 64-year-old male (PromoCell GmbH, Heidelberg, Germany) – were used in this study. Because these cells are commercially available, no patient consent or approval from Ethics Committee was needed.
Cells were cultured at 37°C and 8% carbon dioxide with the media recommended by the manufacturer. The media were changed every other day and the cells were split enzymatically with Trypsin: 1× phosphate-buffered saline (Ref. 14190–185; Gibco Life Technologies) and Tryple Express (Ref. 12604–013; Gibco-Life Technologies) for PT-2501 cells, and the Trypsin kit (PromoCell GmbH) for C-12974 cells. Cultures were trypsinized when the cells were approximately 90% confluent.
Cells were cultured at 37°C and 8% carbon dioxide with the media recommended by the manufacturer. The media were changed every other day and the cells were split enzymatically with Trypsin: 1× phosphate-buffered saline (Ref. 14190–185; Gibco Life Technologies) and Tryple Express (Ref. 12604–013; Gibco-Life Technologies) for PT-2501 cells, and the Trypsin kit (PromoCell GmbH) for C-12974 cells. Cultures were trypsinized when the cells were approximately 90% confluent.
6
3D Bone Mimetic Prostate Cancer Model
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Human MSCs (PT-2501) (Lonza) were maintained in MSCGM™ Bulletkit™ medium (Lonza, PT3001). Human prostate cancer cell line MDA-PCa-2b (PCa) is purchased from ATCC and kept in 80% BRFF-HPC-1 (AthenaES) and 20% FBS (ATCC). For 2D cultures, cells were seeded on tissue culture-treated Petri dishes. For 3D sequential culture, MSCs were seeded at a density of 5 × 104 cells per scaffold and cultured for 23 days to allow bone tissue formation. Further, prostate cancer cells PCa were seeded on newly formed bone tissue in the 3D bone mimetic scaffolds at a density of 5 × 104 cells and maintained in 1:1 MSCs and PCa medium (Supplementary Fig. 4 ).
7
Culturing Human and Porcine BM-MSCs
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Human BM-MSC commercial cell line was obtained from Lonza (Lonza, PT-2501). Porcine BM-MSCs were isolated from transgenic porcine embryos which constitutively expressed green fluorescent protein (GFP). Both human and porcine BM-MSCs were cultured in high-glucose DMEM (Dulbecco’s modified Eagle’s medium, Invitrogen) supplemented with 15% FBS (fetal bovine serum, Invitrogen) and 10 μg/ml gentamycin (Sigma Aldrich) in 5% CO2 at 37 °C. Culture medium was replaced every 2 days, and cells were passaged after reaching confluency. For further analysis, cells from passages 4–9 were used. Depending on the type of analysis, cells were collected and frozen (for further RNA isolation) or cultured on cover slides covered with a 1% gelatin solution and fixed with a 3% PFA (for protein immunolocalization).
8
hMSC Expansion and Comparative Analysis
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Human mesenchymal stem cells (hMSC) were obtained from Lonza (PT-2501) and were expanded in MSCGM for use in comparative flow cytometry and confocal analysis with MLPC.
9
Umbilical Cord Matrix Cell Isolation
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Two to three centimeters of term umbilical cord was purchased from LifeLine Stem Cells (New Haven, IN), and dissected in sterile Hank's Buffered Saline (Gibco, Canada) containing 1% gentamycin using aseptic technique. The vein and arteries were carefully removed. Pieces of Wharton's jelly (umbilical cord matrix) were then transferred to collagenase I and II (1 mg/ml, Gibco), diced into small pieces and incubated for up to 2 hours at 37°C on an orbital shaker. Ca2+ and Mg2+‐free phosphate‐buffered saline (PBS, Gibco) was then added to the viscous cell suspension prior to trituration and centrifugation at 2,000 rpm for 10 minutes at room temperature.
FTM HUCPVCs were isolated as per a published protocol47 and were obtained from the CReATe Fertility Centre (Sunnybrook Research Institute Research Ethics Board Project Identification Number 454‐2011). An in vitro characterization and comparison between term and FTM HUCPVCs had been previously reported 47 . Other cell types used included passage‐matched adult human BMSCs which were obtained commercially (PT‐2501, Lonza, Switzerland). The expression level of CD44 was revalidated for the three cell lines (Supporting Information Fig. S1).
FTM HUCPVCs were isolated as per a published protocol
10
Optimizing Genipin Crosslinking for hMSC Viability
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