Tryple select

Manufactured by Thermo Fisher Scientific

Sourced in United States, Japan, United Kingdom, Germany, Switzerland, Canada

TrypLE Select is a recombinant trypsin-like protease used for the dissociation and detachment of adherent cells in cell culture applications. It is a ready-to-use, serum-free, and animal-component-free solution that effectively enzymatically disaggregates cells without the need for additional supplementation.

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TrypLE select 10x TrypLE™ Select 10× TrypLE™ Select Enzyme 1× Tryple Express Select ×10 TrypLE™ Select CTS™ TrypLE Select Enzyme solution TrypLE Select Enzyme (10×) TrypLE Select Enzyme 10X TrypLE™ select without phenol red Gibco™ TrypLE™ Select

520 protocols using tryple select

Intestinal and Biliary Organoid Culture

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Adult mouse small intestinal organoids or biliary organoids were grown for a minimum of 12 days as 3D organoids under culture conditions listed above. Intestinal organoids were cultured for 4 days on 50% L‐WRN conditioned medium to increase the number of stem cells. Biliary organoids were then dissociated into single cells by incubating in a digestion solution (TrypLE Select (Gibco), 0.5 M EDTA (Sigma‐Aldrich), 10 μM Rock Inhibitor Y‐27632 (R&D Systems) solved in PBS) and seeded onto 48 Well plates (Corning) pre‐coated by 10% Matrigel (Corning) for 20 min. Intestinal organoids were dissociated into single cells by digesting in 10% TrypLE Select (Gibco) and seeded onto 48 Well plates (Corning) pre‐coated by 3,3% Matrigel (Corning) for 2 h (∼20,000 cells were seeded per well). Intestinal 2D monolayer were cultured in IntestiCult Organoid Growth Medium (Human) (STEMCELL Technologies) with added 10 μM Rock Inhibitor Y‐27632 (R&D Systems). Ductal monolayer were cultured in ductal expansion medium. Initially, intestinal organoids were submerged in 150 μL 50% L‐WRN media with 10 μM Rock Inhibitor Y‐27632 (R&D Systems).

Bittel M., Reichert P., Sarfati I., Dressel A., Leikam S., Uderhardt S., Stolzer I., Phu T.A., Ng M., Vu N.K., Tenzer S., Distler U., Wirtz S., Rothhammer V., Neurath M.F., Raffai R.L., Günther C, & Momma S. (2021). Visualizing transfer of microbial biomolecules by outer membrane vesicles in microbe‐host‐communication in vivo. Journal of Extracellular Vesicles, 10(12), e12159.

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Establishment and Maintenance of Human Induced Pluripotent Stem Cells

Cited 2 times

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Two hiPSC lines (201B7 and FF-PB-3AB4) were used in the experiments in this study. The validated hiPSC line 201B7 was purchased from Riken Cell Bank (Tsukuba, Japan) and transferred from on-feeder to feeder-free conditions in our laboratory. The hiPSC line FF-PB-3AB4 was established in our laboratory (Suzuki et al., 2019 (link)). These hiPSC lines were cultured according to a previously described method (Nakagawa et al., 2015 (link)) with slight modifications. In brief, the hiPSCs were maintained in StemFit AK02N medium (Ajinomoto, Tokyo, Japan) with penicillin (50 units/mL) and streptomycin (50 μg/mL) (#1514022; Gibco, Carlsbad, CA, USA) at 37°C with 5% CO₂. The medium was changed every other day and passaged every 7 days using 0.5x TrypLE Select (1x TrypLE Select [#A12859-01; Gibco] diluted 1:1 with 0.5 mM EDTA [#06894-14; Nacalai Tesque, Kyoto, Japan]/PBS [-]) and Rho-associated kinase (Rock) inhibitor (Y-27632; WAKO, Osaka, Japan). iMatrix-511 silk (#892021; Nippi, Tokyo, Japan) was used for precoating.

Uehara K., Koyanagi-Aoi M., Koide T., Itoh T, & Aoi T. (2022). Epithelial-derived factors induce muscularis mucosa of human induced pluripotent stem cell-derived gastric organoids. Stem Cell Reports, 17(4), 820-834.

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Isolation and Adipogenic Differentiation of ASCs

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To purify ASCs, SVF (1.5–2.0 × 10⁷ cells) was plated onto a 150-mm culture dish and cultivated in minimum essential medium alpha (containing nucleosides) supplemented with 10% fetal bovine serum (HyClone, USA)/antibiotic-antimycotic (Gibco, Massachusetts, USA) at 37°C and 5% CO₂ under humid conditions. After culture for 72 h, the nonadherent cells in the dish were completely removed with phosphate-buffered saline (Gibco), and the adherent cells were cultured to subconfluence. Concentrated ASCs (adherent cells) were collected using TrypLE Select (Gibco).
To prepare adipocytes, ASCs were induced to adipogenic differentiation using a StemPro Adipogenesis Differentiation Kit (Gibco). The confluent culture of ASCs was incubated under adipogenic conditions for 14 d. After detachment by TrypLE Select (Gibco), floating cells (adipocytes) were separated from the precipitate (immature cells) by centrifugation, and the adipocytes were collected using micropipettes.

Nishimura A., Kumagai T., Nakatani M, & Yoshimura K. (2016). Method for selective quantification of adipose-derived stromal/stem cells in tissue. Journal of Biological Methods, 3(4), e58.

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Flow Cytometry Analysis of Cell Cycle and Pluripotency

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For the cell cycle analysis, cells in the DNA synthesis phase were labeled with EdU using Click-iT™ Plus EdU Flow Cytometry assay kits (Thermo Fisher Scientific, Waltham, MA, USA), and the DNA was stained using a FxCycle™ PI/RNase staining solution (Thermo Fisher Scientific, Waltham, MA, USA). Then, 47 h after Frk or Dex stimulation, the EdU was added and labeled for 1 h. Cells were harvested using TrypLE Select (Thermo Fisher Scientific, Waltham, MA, USA), fixed with 4% paraformaldehyde, and conjugated with an Alexa Fluor ™ 488 dye to detect EdU. Then, PI staining was performed and analyzed on a FACSVerse flow cytometer (BD Biosciences, San Jose, CA, USA). For the evaluation of the pluripotency of hiPSCs, cells were harvested using TrypLE Select and resuspended in PBS containing 1% bovine serum albumin (BSA); then they were incubated with an anti-human SSEA-4 (Thermo Fisher Scientific, Waltham, MA, USA) or anti-mouse IgG3 isotype control (Thermo Fisher Scientific, Waltham, MA, USA) for 45 min on ice. Next, the cells were washed and resuspended in PBS containing 1% BSA. SSEA-4 positive cells were analyzed on a FACSVerse flow cytometer. Data were analyzed using the FACSuite software (BD, Biosciences, San Jose, CA, USA).

Kaneko H., Kaitsuka T, & Tomizawa K. (2020). Response to Stimulations Inducing Circadian Rhythm in Human Induced Pluripotent Stem Cells. Cells, 9(3), 620.

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Maintenance and Differentiation of Human iPSCs and Neural Cells

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The hiPSC line 1210B2 was maintained as described previously^{51 (link),52 (link)}. The hiPSCs were dissociated using 0.5× TrypLE Select (Thermo Fisher) and plated on iMatrix511 (Nippi)-coated dishes in StemFit (AK03N) medium at feeder-free condition. The hiPSC-derived NECs were established and maintained as described previously^{53 (link),54 (link)}. The hNECs were passaged every 4 days and plated at a ratio of 1:5. The cells were dissociated using TrypLE Select (Thermo Fisher) and plated on Matrigel (Corning)-coated dishes in RHB-A medium (Takara) supplemented with 10 ng/mL EGF (Peprotech) and 10 ng/mL FGF2 (Peprotech). The hiPSC-derived NCCs were established previously and passaged less than three times for analysis. The hNCC medium consisted of 1:1 neurobasal medium (Thermo Fisher Scientific) and D-MEM/Ham’s F-12 (Wako) medium containing 1× GlutaMax (Thermo Fisher Scientific), 0.5× GEM 21 NeuroPlex serum-free supplement (Gemini Bio Products), 0.5× N2 supplement (Thermo Fisher Scientific), 5 mg/mL insulin (Sigma-Aldrich), 0.5% penicillin and streptomycin (Nacalai tesque), 20 ng/mL FGF2 and 20 ng/mL EGF. The hNCCs were dissociated using TrypLE Select and plated on fibronectin (Sigma-Aldrich)-coated dishes.

Sanosaka T., Okuno H., Mizota N., Andoh-Noda T., Sato M., Tomooka R., Banno S., Kohyama J, & Okano H. (2022). Chromatin remodeler CHD7 targets active enhancer region to regulate cell type-specific gene expression in human neural crest cells. Scientific Reports, 12, 22648.

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CRISPR/Cas9-mediated gene editing in human iPSCs

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HA-iPSCs cultured on Matrigel were detached with TrypLE Select (Life Technologies, Grand Island, NY, USA). The dissociated single cells were resuspended with 100 μL of Human Stem Cell Nucleofector Kit 2 (Lonza,) and nucleofected using Nucleofector II (Lonza) set at program B016. For 1 × 10⁶ cells, 2.5 μg of each CRISPR/Cas9 plasmid was used, with or without 50 pmol ssODN synthesized by Sangon Biotech (Shanghai, China). The transfected cells were seeded on Matrigel-coated wells in mTeSR1 (Stem Cell Technologies, Vancouver, BC, Canada) containing 10 μM Y27632. After 2 days, the cells were detached with TrypLE Select (Life Technologies), and 10,000 cells were seeded on MEF feeders in ESC medium containing 10 μM Y27632 in a 100-mm culture dish.⁶² (link) Approximately 2 weeks later, clones were picked and expanded. To detect gene deletion events, DNA from individual colonies was extracted and subjected to PCR. PCR was performed using the primers F8-E14-forward (F): 5′-GGTGGACCTCTGAGCTTGAGTGA-3′ and F8-E14-reverse (R): 5′-GCATCTTAAAGAACGACATATCTGGAT-3′. The PCR products were identified by agarose gel electrophoresis. The 448-bp product was sequenced by Sanger sequencing.

Hu Z., Zhou M., Wu Y., Li Z., Liu X., Wu L, & Liang D. (2019). ssODN-Mediated In-Frame Deletion with CRISPR/Cas9 Restores FVIII Function in Hemophilia A-Patient-Derived iPSCs and ECs. Molecular Therapy. Nucleic Acids, 17, 198-209.

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Oxidative Status, Lipid Accumulation, and oxLDL Uptake in C-MSCs

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To evaluate C‐MSC oxidative status, cells cultured in basal medium were incubated for 30 min with 10 μM dichlorofluorescein (Sigma‐Aldrich, St. Louis, Missouri, USA) and detached with TrypLE Select (Life Technology, Carlsbad, California, USA), and the conversion into the fluorescent dye 2′,7′‐DCF by cell ROS was measured by flow cytometry (Gallios, Beckman Coulter, Brea, California, USA). The mean FITC fluorescence was measured.
To determine the correlation between CD36 expression and lipid accumulation, cells were stained using 12.5 ng/ml Nile Red (Invitrogen, Carlsbad, California, USA), to mark intracellular neutral lipids, and 2.5 μl of anti‐CD36 antibody (Life Technologies, Carlsbad, California, USA). The mean of the fluorescence was determined for Nile Red and CD36 for each sample.
To quantify the DiI‐oxLDL internalization, cells were treated with 10 μg/ml DiI‐oxLDL for 3 h, detached with TrypLE Select (Life Technologies, Carlsbad, California, USA), and acquired with FACS Gallios (Beckman Coulter, Brea, California, USA). The mean APC fluorescence was determined for each sample.

Sommariva E., Stadiotti I., Casella M., Catto V., Dello Russo A., Carbucicchio C., Arnaboldi L., De Metrio S., Milano G., Scopece A., Casaburo M., Andreini D., Mushtaq S., Conte E., Chiesa M., Birchmeier W., Cogliati E., Paolin A., König E., Meraviglia V., De Musso M., Volani C., Cattelan G., Rauhe W., Turnu L., Porro B., Pedrazzini M., Camera M., Corsini A., Tondo C., Rossini A, & Pompilio G. (2021). Oxidized LDL‐dependent pathway as new pathogenic trigger in arrhythmogenic cardiomyopathy. EMBO Molecular Medicine, 13(9), e14365.

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Feeder-free culture of iPSC line 201B7

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The validated iPSC line 201B7 was purchased from Riken Cell bank (Tsukuba, Japan) and transferred from on-feeder to feeder-free conditions in our laboratory. We cultured the iPSC lines according to a previously described method (Nakagawa et al., 2014 (link)). In brief, the culture plates were precoated with iMatrix-511 (0.5 μg/cm²), and the iPSC were maintained in Stem Fit medium (Ajinomoto) with penicillin (100 units/mL) and streptomycin (100 μg/mL; Life Technologies, MA, USA) at 37°C with 5% CO₂. The medium was changed every other day and passaged every 7-10 days using 0.5× TrypLE Select (1× TrypLE Select diluted 1:1 with 0.5 mM EDTA/PBS [-]; Life Technologies) and Rho-associated kinase (Rock) inhibitor (Y-27632; WAKO, Osaka, Japan).

Koide T., Koyanagi-Aoi M., Uehara K., Kakeji Y, & Aoi T. (2022). CDX2-induced intestinal metaplasia in human gastric organoids derived from induced pluripotent stem cells. iScience, 25(5), 104314.

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Expansion and Characterization of MSCs

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Cells were cultured in a 5% CO₂, 37°C incubator in MSC CCM on appropriately sized t-flasks. At 24 hours and 48 hours after seeding, non-adherent cells were removed via media change. In these culture conditions, only mesenchymal stem cells from the mononuclear cell population adhered to the surface. Cell enumeration and characteristics (doubling time, viability, senescence, and apoptosis) taken on and after the first harvest reflect only MSC characteristics. Media changes were performed every 2–4 days until cells reached the desired 70–80% confluence between days 8–12. When cells reached desired 70–80% confluence, they were passaged. MSCs were washed with Dulbeccos phosphate buffered saline (DBPS, Invitrogen, Grand Island, NY), removed from the culture surface with TrypLE select® (Invitrogen, Grand Island, NY), diluted with MSC CCM to quench the action of TrypLE select®, and centrifuged. Cells were resuspended in MSC CCM and analyzed for viability, senescence, and apoptosis. Half of the remaining cells that were not used for assays were seeded in new flasks at densities of 40–50 cells/cm², and the rest were frozen down at each passage. A total of 6 passages were performed, which corresponds to approximately 35–40 population doublings in healthy cells.

Pollock K., Sumstad D., Kadidlo D., McKenna D.H, & Hubel A. (2014). Clinical mesenchymal stem cell products experience functional changes in response to freezing. Cytotherapy, 17(1), 38-45.

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Flow Cytometric Analysis of ECFCs

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Flow cytometric analysis (FACS) of isolated ECFCs was performed as previously described.15 Briefly, cells were dissociated using 1X TrypLE select (Invitrogen) and washed once with the FACs buffer with 10% FBS, followed by an additional wash with FACs buffer. A list of the antibodies used can be found as Online Supplemental data. The samples were analyzed with the MACSQuant VYB (Miltenyi, Bergisch Gladbach, Germany) with the following instrument settings: Blue/488 FITC, A488: 525/50; Yellow/561 PE: 586/15, APC: 661/20, APC‐Cy7: 750 LP.

Sánchez‐Duffhues G., Williams E., Benderitter P., Orlova V., van Wijhe M., Garcia de Vinuesa A., Kerr G., Caradec J., Lodder K., de Boer H.C., Goumans M., Eekhoff E.M., Morales‐Piga A., Bachiller‐Corral J., Koolwijk P., Bullock A.N., Hoflack J, & ten Dijke P. (2019). Development of Macrocycle Kinase Inhibitors for ALK2 Using Fibrodysplasia Ossificans Progressiva‐Derived Endothelial Cells. JBMR Plus, 3(11), e10230.

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