Cells were isolated and expanded on the Quantum Cell Expansion System (Terumo BCT, Lakewood, CO) as described (Cunha et al., 2017 (link)) in off-the-shelf xenobiotic free media supplemented with additional MAPC growth components, maintained under low oxygen tension, and stored in liquid nitrogen gas phase until use. Cells were phenotyped in terms of marker expression and differentiation potential, as described (Crabb é et al., 2016 (link)). In short, MAPCs were phenotyped by their ability to differentiate into adipocytes, chondrocytes and osteoblasts, flow cytometry (CD13+, CD90+, CD105+, CD45-, HLAII-, CD34-), expression of key MAPC markers miR-204-5p, miR-20a-5p, miR-18a-5p, miR-106a-5p, miR-17-5p, and miR-155-5p and no expression for seven key MSC miRNAs (miR-335-5p, miR-145-5p, miR-143-3p, miR-27b-3p, miR-125b-5p, miR-26a-5p, miR-152-3p). Proangiogenic activity was measured by tube formation on human umbilical vein endothelial cells and immunosuppressive capacity by their ability to reduce the proliferation of human, third party CD3/28 activated T-cells. MAPCs (1 × 107 cells/mL) were thawed at room temperature for maximally 1 h and sterile administered in a single 1 mL bolus via the femoral vein to the fetuses (127 GA) randomized to MAPC treatment.
Quantum cell expansion system
Manufactured by Terumo BCT
Sourced in United States
The Quantum Cell Expansion System is a laboratory equipment designed for the controlled expansion and culture of cells. It provides a closed, automated, and sterile environment for the growth and maintenance of various cell types, including stem cells, immune cells, and other cell populations of interest.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin p s, by Thermo Fisher Scientific (1 mentions)
Human fibronectin, by Corning (1 mentions)
α mem, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Alpha mem, by Thermo Fisher Scientific (1 mentions)
Cryostor, by BioLife Solutions (1 mentions)
8 protocols using quantum cell expansion system
1
MAPC Isolation, Expansion, and Therapeutic Administration
2
Quantum Cell Expansion System Protocol
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The Quantum™ Cell Expansion System (Quantum™ system; Terumo BCT, Inc., Lakewood, CO, USA) is a functionally closed hollow-fiber bioreactor (HFBR) system enclosed in a stand-alone incubator. It is designed for culturing both adherent and suspension cells. The surface area is 1.7 m2, which contains approximately 9200 hollow fibers (which is the equivalent of 100 T-flasks of 175 cm2) (Figure 1 ). The Quantum system fluid circuit is designed around two fluid loops for the intracapillary (IC) and extracapillary (EC) sides of the bioreactor. The bioreactor membrane allows free gas diffusion between the IC and EC sides of the bioreactor, as well as small molecule diffusion. Larger macromolecules are sequestered on the side of the membrane where they are added. The process, medium perfusion rate, harvest time, incubation time, and other tasks are controlled and adjusted by the user via a touchscreen surface.
3
Umbilical Cord MSC Expansion under Hypoxia
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Umbilical cords (UCs) were collected with ethical approval (National Research Ethics Service; 10/H10130/62), and research was carried out according to the Helsinki Declaration. MSCs were isolated from the whole umbilical cord, as previously described [13 (link)]. UCMSCs were first cultured expanded in a Quantum® Cell Expansion System (n = 4) (Terumo BCT, Surrey, UK) to passage 1 and then grown on plastic thereafter. UCMSCs were grown under normoxic conditions (21% O2) and hypoxic conditions (5% O2). UCMSCs were fed every 2–3 days with DMEM F12, 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (P/S) (Life Technologies, Warrington, UK). The oxygen content of the DMEM F12 was lowered to approximately 5% using the HypoxyCOOL™ media conditioning system (Baker Ruskinn, Bridgend, UK) for 3 h before adding to cells into the InvivO2 hypoxic workstation (Baker Ruskinn, Bridgend, UK). At 80% confluence, cells were washed with PBS, and DMEM F12 with 10% FBS (EV-depleted) was added for 48 h. Population doubling times (PDT) were calculated using the formula DT = T ln2/ln(Xe/Xb), where T is the incubation time, Xb is the cell number at the beginning of the incubation time, and Xe is the cell number at the end of the incubation time.
4
Expansion of Adipose-Derived MSCs
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Starting from the lipoaspirate of a donor, the AT-MSCs were expanded in flasks until a number of at least 20 × 106 cells was reached for each passage, not exceeding P3. In order to produce a high amount of cells for in vivo experiments, the MSCs were expanded using the bioreactor Quantum Cell Expansion System (Terumo BCT Inc., Lakewood, CO, USA) and GMP-compliant reagents, as previously described [23 (link)]. Briefly, after priming of the disposable expansion set (the bioreactor was coated overnight with 5 mg of human fibronectin (Corning Incorporated, Deeside, UK)) to promote cell adhesion, a 4 L media bag was then attached to the appropriate inlet line on the Quantum disposable expansion set. The expanded MSCs were analyzed for the expression of the typical MSCs markers (CD90, CD73, CD105) by using monoclonal antibodies (Becton Dickinson, Franklin Lake, NJ, USA), as previously described [22 (link)].
5
LM-NSC008 Cell Expansion and Intranasal Delivery
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Generation and characterization of LM-NSC008 cells stably expressing L-MYC has been previously described [41 (link)]. Cells were passaged and characterized for up to passage 50 [42 (link)]. Briefly, LM-NSC008 cells were expanded to master cell banks using the Quantum Cell Expansion System (Terumo BCT) and frozen at a concentration of 2.4 × 107 cells in CryoStore (BioLife Solutions) in liquid nitrogen. LM-NSC008 cells were thawed, washed with PBS, and administered via IN drops into rats bearing CCI or sham injury using previously optimized protocols [25 (link), 43 (link)].
6
Isolation and Characterization of MAPC Cells
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Human MAPC cells were isolated by Athersys/Regenesys (Athersys, Cleveland, OH, USA; Regenesys, Heverlee, Belgium) from bone marrow of healthy volunteers. A research-grade variant of the clinical product was used: cells were cultured under the same medium conditions as MultiStem®, but not in a Good Manufacturing Practice (GMP) environment. Isolation and cultivation of the MAPC cells were based on previously published protocols [19 (link), 23 (link)]. The Quantum Cell Expansion System (Terumo BCT, Lakewood, CO, USA) was used for ex vivo expansion of large batches of MAPC. All cell batches were subjected to several quality control assays to test if all MAPC cell criteria were met. First of all, cell quality is assessed by measuring viability and plating efficiency post-thawing. Cells were thawed at a population doubling of 29.4 and had a post-thawing viability of ≥ 96%. Second, cells are identified using qPCR and flow cytometry to test both negative and positive markers [15 (link), 24 (link)]. A tube formation assay is done to define the proangiogenic activity [25 (link)], a CFSE assay [26 (link)] is performed for evaluating the immunoregulatory capacity.
7
Isolation and Expansion of Human Bone Marrow MSCs
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Mesenchymal stem cells from human bone marrow were obtained from Lonza (Walkerville, MD, USA). Human bone marrow mesenchymal stem cells (MSC) were cultured in α-MEM from Sigma-Aldrich (Oakville, ON, Canada) with 10% fetal bovine serum from Life Technology (Burlington, ON, Canada) and 2 mM glutamax from Thermo Scientific (Rockford, IL, USA). A master bank was prepared by expanding the cells for 7 days using a Quantum Cell Expansion System from Terumo BCT (Lakewood, CO, USA). The MSC population was homogenuous, with >95% of the cells of phenotype CD73+CD90+CD105+, as determined by flow cytometry [55 (link)].
8
Autologous Adipose-Derived Mesenchymal Stem Cell Protocol
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The AT-MSC product was manufactured by Cardiology Stem Cell Centre (CSCC), University Hospital Copenhagen, under a Manufacturing and Importation Authorization granted by the Danish Medicines Agency. Lipoaspirates were obtained from three healthy consenting female donors (22–26 years old) according to an established protocol26 (link)–28 (link). Cells were isolated from lipoaspirates by enzymatic digestion with collagenase, and the AT-MSCs were expanded for two passages in automated closed bioreactor systems (Quantum Cell Expansion System, Terumo BCT) with 5% human platelet lysate as the growth supplement (Sexton Biotechnologies) in MEM alpha (Gibco) and Penicillin/Streptomycin (Gibco). The AT-MSCs were cryopreserved at 50 × 106/mL CryoStor (BiolifeSolutions) in CellSeal vials (Sexton Biotechnologies) and stored at <−180 °C in nitrogen dry-storage until clinical use. Authorization of tissue establishment for the handling of human tissues and cells has been licensed by The Danish Patient Safety Authority. The test group received ultrasound-guided injections of 25 × 106 AT-MSCs bilaterally in the submandibular glands and 50 × 106 AT-MSCs in each parotid gland between April 2020 and January 2021 at Rigshospitalet, University Hospital of Copenhagen, Denmark.
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