Stemmacs msc expansion media xf

Manufactured by Miltenyi Biotec

Sourced in Germany

StemMACS MSC Expansion Media XF is a serum-free and animal component-free medium specifically designed for the expansion of mesenchymal stem/stromal cells (MSCs) in vitro. It supports the long-term growth and maintenance of MSCs without altering their differentiation potential.

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StemMACS™ MSC Expansion Media Kit XF StemMACS MSC Expansion Media StemMACS expansion media StemMACS media

7 protocols using stemmacs msc expansion media xf

Isolation and Culture of Umbilical Cord-Derived Mesenchymal Stem Cells

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The umbilical cord was collected directly after birth in 0.9% sodium chloride
(Bidiphar, Vietnam) and transferred to a cell processing center. UC-MSCs were
isolated under xeno-free and serum-free conditions as described previously^{12 (link)}. Briefly, the cord was washed in phosphate buffered saline (PBS) and 70%
alcohol and then cut into small pieces. The mixture was digested in 500 U/ml
collagenase (Gibco, Grand Island, NY, USA) at 37°C using a GentleMACS
Dissociator (Miltenyi, Germany). Cells were filtered and suspended in PowerStem
MSC1 Medium (PAN Biotech, Germany) (donor 1) or StemMACS™ MSC Expansion Media XF
(Miltenyi, Germany) (donor 2) supplemented with 100 U/ml Pen/Strep (Life
Technologies, USA). They were seeded in treated cell culture flasks (NUNC Thermo
Scientific, Rochester, NY, USA), coated with CellStart™ substrate (Thermo Fisher
Scientific, Grand Island, NY, USA) and cultured at 37°C under 5% CO₂.
The cells were harvested once they reached 80% confluency. The cells were
passaged for subsequent culture at a cell density of 4,000 cells/cm²and quality characterization or cryopreserved in CryoStor® CS10 (Stem Cell
Technology, Canada) in the gas phase of liquid nitrogen in an automated Brooks
System (Brooks Life Science, Chelmsford, MA, USA) to maintain the temperature at
–196°C.

Nguyen Thanh L., Hoang V.T., Le Thu H., Nguyen P.A., Hoang D.M., Ngo D.V., Cao Vu H., Nguyen Thi Bich V, & Heke M. (2022). Human Umbilical Cord Mesenchymal Stem Cells for Severe Neurological Sequelae due to Anti-N-Methyl-d-Aspartate Receptor Encephalitis: First Case Report. Cell Transplantation, 31, 09636897221110876.

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Expansion of Human Mesenchymal Stem Cells

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Human mesenchymal stem cells from adipose (AD), amniotic (AM), bone marrow (BM), chorionic (CH), liver (LI), and umbilical cord matrix (UC) tissues were obtained frozen (Sciencell, Carlsbad, CA; ATCC, Manassas, VA; Promocell, Heidelberg, Germany). Cells were seeded at a density of 5,000 cells per cm² and cultured for two to three passages in T-flasks with human StemMACS MSC Expansion Media XF (Miltenyi Biotec, Bergisch Gladbach, Germany) (which was serum- and xenobiotic-free), including manufacturer-supplied supplement, 100 units/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL fungizone (Gibco/Thermo Fisher Scientific, Pittsburgh, PA). Cell viabilities and numbers were monitored by Trypan Blue exclusion in a Neubauer Chamber.

Schmelzer E., McKeel D.T, & Gerlach J.C. (2019). Characterization of Human Mesenchymal Stem Cells from Different Tissues and Their Membrane Encasement for Prospective Transplantation Therapies. BioMed Research International, 2019, 6376271.

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Isolation and Culture of Mesenchymal Stem Cells

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Freshly detached MPCs obtained as above were seeded at 2x10⁴ cells/cm² in TC-treated T25 flasks and let to adhere in DMEM/10% PhABS. After 24 h medium was replaced with StemMACS^™ MSC Expansion Media XF (Miltenyi Biotec) and cultures grown to confluence to obtain passage one MSCs (P1-MSCs). Cells were then detached with TrypLE Select^® and sub-cultured 1:2 to confluence to obtain passage two MSCs (P2-MSCs). In parallel, 6-well plates were set up in duplicate to draw corresponding growth curves. AlamarBlue reduction assay was performed at day 7 and day 14 adding 10% PrestoBlue^® Cell viability assay (Thermo Fisher) to culture medium. After 8 h incubation, 100 μl of culture medium were harvested and absorbance was measured at 570 and 600 nm by Benchmark Plus microplate spectrophotometer (BioRad, Hercules, USA-CA). Percentage of reduced AlamarBlue was calculated according to manufacturer and two-tailed unpaired t-test was performed. Growth curves were also acquired after cell treatment with 0.5 μM calmidazolium chloride (CLMDZ) (Sigma Aldrich, St. Louise, USA-MO) or 2 and 3 nM bortezomib (BTZM) (Selleckchem, Houston, USA-TX).

Pacini S., Montali M., Mazziotta F., Schifone C.P., Macchia L., Carnicelli V., Panvini F.M., Barachini S., Notarfranchi L., Previti G.B., Buda G, & Petrini M. (2019). Mesangiogenic progenitor cells are forced toward the angiogenic fate, in multiple myeloma. Oncotarget, 10(63), 6781-6790.

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Mesenchymal Stem Cell Differentiation

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Freshly detached cells from primary cultures were replated at 20,000 cells/cm² and let adhere in DMEM/10% PhABS for 24 h. Culture medium was then replaced with StemMACS^® MSC Expansion Media XF (Miltenyi Biotec), and cells cultured up to 80% of confluence (usually 7–8 days) to obtain P1-MSCs. Cultures were then incubated for further 7–8 days to complete mesengenic differentiation (P2-MSCs). Cell osteogenic and adipogenic potential was tested. P2-MSCs were replated at 20,000 cells/cm² in TC-treated 6-wells plates and grown to confluence. Medium was then replaced with either StemMACS^® OsteoDiff Media, StemMACS^® AdipoDiff Media, or expansion medium (negative controls). Two to 3 weeks later, calcium deposits were revealed by staining with alizarin S (Sigma Aldrich) and lipid droplets revealed by staining with Nile red 200 nM (Thermo Fisher Scientific), according to manufacturer’s. Imaging was performed on inverted fluorescence DM IRB Leica microscope (Leica, Wetzlar, Germany), equipped with LAS image acquisition software (Leica).

Barachini S., Montali M., Panvini F.M., Carnicelli V., Gatti G.L., Piolanti N., Bonicoli E., Scaglione M., Buda G, & Parchi P.D. (2021). Mesangiogenic Progenitor Cells Are Tissue Specific and Cannot Be Isolated From Adipose Tissue or Umbilical Cord Blood. Frontiers in Cell and Developmental Biology, 9, 669381.

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Isolation and Characterization of Healthy Donor Mesenchymal Stromal Cells

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Human mesenchymal stromal cells from healthy donors (HD-hMSCs) were derived from BM aspirates. In detail, 1 × 10⁶ BM cells were plated (40 000 cells per cm²) in StemMACS (MSC Expansion Media XF, Miltenyi Biotech, BergischGladbach, Germany) medium supplemented with 100 U/mL penicillin/streptomycin (Gibco, Life Technologies, CA) and incubated at 37°C. The adherent cells were expanded to 90% confluence in T75 flasks before their use. HD-hMSCs were trypsinized (Trypsin/EDTA phosphate-buffered saline 1:250 Solution, BioConcept, Allschwill, Switzerland) at 37°C for 5 minutes, washed with Hanks balanced salt solution (BioConcept, Allschwill, Switzerland), and resuspended in an appropriate medium. HD-hMSCs between passages 3 to 5 were used. Immunophenotype analysis (hCD45-ECD-Beckman Coulter; hCD29-PE and hCD73-PE, Thermo Fisher Scientific) and osteogenic/adipocyte differentiation assay (PromoCell, Heidelberg, Germany) were carried out to characterize derived HD-hMSCs before use (supplemental Figure 1A-B). Project ID 281/2015-PR was approved by the local Ethical Committee of Padua University. The study was conducted following the Declaration of Helsinki.

Cani A., Tretti Parenzan C., Frasson C., Rampazzo E., Scarparo P., Francescato S., Caicci F., Barbieri V., Rosato A., Cesaro S., Zecca M., Micalizzi C., Sainati L., Pigazzi M., Biffi A., Buldini B., Locatelli F., Persano L., Masetti R., te Kronnie G, & Bresolin S. (2022). Long-term proliferation of immature hypoxia-dependent JMML cells supported by a 3D in vitro system. Blood Advances, 7(8), 1513-1524.

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Isolation and Expansion of Mesenchymal Stem Cells

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Bones were broken into fragments and incubated for 1 h at 37 °C in phosphate-buffered saline (PBS) supplemented with 1 mg/mL collagenase type I (Thermo Fisher, Waltham, MA, USA). Supernatants were filtered in a cell strainer with 100 µm nylon mesh pores (Greiner Bio-One, Kremsmünster, Austria). Afterwards, bone fragments retained in the cell strainer were transferred into StemMACS MSC Expansion Media XF (Miltenyi Biotec, Bergisch Gladbach, Germany) supplemented with 1% penicillin/streptomycin. Then, adherent MSC were expanded in T175 flasks in a humidified 5% CO₂ atmosphere at 37 °C and passaged at 80% confluency.

Kohl V., Fabarius A., Drews O., Bierbaum M., Jawhar A., Darwich A., Weiss C., Flach J., Brendel S., Kleiner H., Seifarth W., Hofmann W.K, & Popp H.D. (2021). Genotoxic Bystander Signals from Irradiated Human Mesenchymal Stromal Cells Mainly Localize in the 10–100 kDa Fraction of Conditioned Medium. Cells, 10(4), 827.

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Isolation and Expansion of MDS-derived MSCs

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MDS patient samples were obtained from diagnostic BM aspirations (BMAs) after informed written consent and in accordance with the Declaration of Helsinki. Mononuclear cells (MNCs) were isolated using Ficoll-Paque (Cytiva, Freiburg, Germany) density gradient centrifugation. CD34+ cell enrichment from MNCs was performed using MACS columns (Miltenyi Biotec, Bergisch Gladbach, Germany). Patient MSCs were selected by plastic adherence and expanded ex vivo with StemMACS MSC Expansion Media XF (Miltenyi Biotec).

Schmitt N., Jann J.C., Altrock E., Flach J., Danner J., Uhlig S., Streuer A., Knaflic A., Riabov V., Xu Q., Mehralivand A., Palme I., Nowak V., Obländer J., Weimer N., Haselmann V., Jawhar A., Darwich A., Weis C.A., Marx A., Steiner L., Jawhar M., Metzgeroth G., Boch T., Nolte F., Hofmann W.K, & Nowak D. (2021). Preclinical evaluation of eltrombopag in a PDX model of myelodysplastic syndromes. Leukemia, 36(1), 236-247.

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