For cell surface marker detection, TrypLE Select (1×) (Thermo Fisher Scientific, cat.no. 12563011) was used for cell dissociation. Cells (3 × 105) were re-suspended in 100 μL of fluorescence-activated cell sorting (FACS) buffer (PBS contained 2% of FBS) and incubated with primary antibodies and appropriate secondary antibodies on ice for 30 min each. Antibodies used in this study are described in Table S3 . The samples were washed with 1 mL of FACS buffer two times and analyzed using a BD LSRFortessa Cell Analyzer (BD Biosciences) with BD FACSDiva software (BD Biosciences). Flow cytometry data were analyzed and generated by FlowJo software (Tree Star, v.9.9.6). The boundaries between positive and negative were defined using un-stained samples or non-reprogrammed MEFs as negative controls.
Tryple select 1
Manufactured by Thermo Fisher Scientific
Sourced in United States
TrypLE™ Select (1×) is a cell dissociation reagent used for the detachment of adherent cells from cell culture surfaces. It is a recombinant trypsin-like protease that effectively dissociates cells while maintaining cell viability and surface antigen expression.
Automatically generated - may contain errors
Lab products found in correlation
Hdpscs, by Lonza (2 mentions)
Lsrfortessa cell analyzer, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Tryple express, by Thermo Fisher Scientific (1 mentions)
Cryostor cs10, by Merck Group (1 mentions)
100 mm cell strainer, by BD (1 mentions)
Cellbind flasks, by Corning (1 mentions)
Evos cell imaging system, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Hdpscs, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Facsaria, by BD (1 mentions)
Facscanto, by BD (1 mentions)
Stemmacs osteodiff media, by Miltenyi Biotec (1 mentions)
Aprotinin, by Merck Group (1 mentions)
Ultra low adherent culture dishes, by Corning (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
7 protocols using tryple select 1
1
Cell Surface Marker Detection Protocol
2
Establishment and Maintenance of KOSM4 hiPSC Line
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The KOSM4 hiPSC line, established using a SeVdp-iPS vector14 (link),15 (link), was used for all experiments unless otherwise stated. The cells were plated on a fibronectin-coated dish with serum-free medium and were maintained in an ESF9a-based medium with daily medium changes14 (link). ESF9a medium consisted of F7 medium (Table S1 ) with 10 μg/mL insulin (19278-5ML, Sigma-Aldrich), 10 ng/mL basic fibroblast growth factor (rhFGF-2, D2222, Katayama Chemical TTD.), and 2 ng/mL activin A (GFH6-1000, R&D Systems). When the cells reached 80% confluence, they were passaged using TrypLE Select (1 ×) (12563-011, Thermo Fisher Scientific) or TrypLE Express (12604-013, Thermo Fisher Scientific).
3
Isolation and Culture of Primary Adherent Cells
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Waste LPI fractions were received from the SNBTS islet isolation lab. LPIs were washed once in SMPL medium, centrifuged at 300 £ g for 5 min and then cultured at 0.006 mL/cm 2 at 37°C in 5% CO 2 in SMPL medium (e.g., 0.45 mL LPI fraction in a T-75 flask plus 9.5 mL SMPL medium). Explant outgrowth was assessed, and adherent cells were observed migrating from the explanted materials. The medium was carefully exchanged at day 7 and thereafter changed every 3À4 days. Cultures were observed and photographed using an EVOS cell imaging system (Thermo Fisher Scientific). Once the cultures had reached 80À90% confluence, the cells were recovered with a 10-min incubation at 37°C with 0.13 mL/cm 2 TrypLE Select £1 (Thermo Fisher Scientific). To remove cell debris, the material was passed through a 100-mm cell strainer (Falcon). The cells were counted using a hemocytometer and designated passage 0. These cells were either cryopreserved at 1 £ 10 6 per 2-mL cryovial in CryoStor CS10 (Sigma-Aldrich) or re-cultured at a density of 3000 cells/cm 2 in CellBIND flasks (Corning).
4
hDPSCs Scaffold Seeding and Culture
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The hDPSCs (Lonza, Walkersville, MD, USA) were cultured and expanded with Alpha-minimum eagle’s medium (α-MEM) supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and were supplemented with 1% v/v penicillin-streptomycin (P/S) in physiological conditions held constant (37 °C air temperature, 5% CO2 atmosphere). The mediums were exchanged every 3 days. Subculturing was conducted when samples reached 70%–80% confluency through dissociation induced with TrypLE™ Select (1×) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). Adherent cells on flask-bottoms were separated by 1.5 mL trypsin-EDTA (0.05% trypsin/0.02% EDTA, Life Technologies Co., Waltham, MA, USA) for 5 min at 37 °C in an incubator.
hDPSCs were prepared into cell suspensions and we adjusted cell concentrations to 1 × 106 cells /mL. C-TCP, 3D-PLGA/TCP, and 3D-TCP (N = 8) samples were placed in a 24-well plate, and 15 μL of hDPSCs suspension was seeded onto each cubic disk for pre-culturing for 2 h. After cells were dropped to the scaffold, culture mediums were refilled.
hDPSCs were prepared into cell suspensions and we adjusted cell concentrations to 1 × 106 cells /mL. C-TCP, 3D-PLGA/TCP, and 3D-TCP (N = 8) samples were placed in a 24-well plate, and 15 μL of hDPSCs suspension was seeded onto each cubic disk for pre-culturing for 2 h. After cells were dropped to the scaffold, culture mediums were refilled.
5
Single-cell Isolation and Antibody Staining
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Single-cell suspensions of EpiSCs and hESCs were obtained using either trypsin-EDTA or TrypleSelect 1 × (Gibco), fixed, stained with the appropriated primary and secondary antibodies according to the manufacturer's protocols and were either analysed with a FACS Canto (Becton Dickinson) or sorted with a FACSAria (Becton Dickinson).
6
Bioprinting hDPSCs for Odontogenic Differentiation
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The hDPSCs (Lonza, Walkersville, MD, USA) were cultured with StemMACS™ Expansion Media XF (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) supplemented with 1% v/v penicillin-streptomycin (P/S) in physiological conditions (37°C, 5% CO2). The medium was exchanged every 3 days. Subculturing was conducted at 70%–80% confluency by dissociation with TrypLE™ Select (1×) (Gibco). StemMACS OsteoDiff media (Miltenyi Biotec GmbH) containing 1% v/v P/S was used as odontogenic differentiation medium. In the culture of hDPSC-laden bio-ink, aprotinin (Sigma) was added to the culture medium at a concentration of 10 μg/mL in order to prevent excessive bio-ink degradation.
For the bioprinting process, harvested hDPSCs were mildly and homogeneously mixed with melted bio-ink at a desired concentration. The prepared hDPSC-laden bio-inks were loaded into 1-mL syringes and incubated in a 4°C refrigerator for 4 min to induce gelation. The bio-ink-laden syringes were connected to the micro-nozzle and installed into the dispensing module of the bioprinting system (Figure 1(a) ).
For the bioprinting process, harvested hDPSCs were mildly and homogeneously mixed with melted bio-ink at a desired concentration. The prepared hDPSC-laden bio-inks were loaded into 1-mL syringes and incubated in a 4°C refrigerator for 4 min to induce gelation. The bio-ink-laden syringes were connected to the micro-nozzle and installed into the dispensing module of the bioprinting system (
7
Seeding hADSCs on Microcarriers
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hADSCs were seeded and incubated at 37 °C in a humidified atmosphere containing 5% CO2. After cells reached 80% confluence, they were harvested by TryplE™ select (1×) (Gibco). Cells were seeded at the density of 104 cells per mg of each type of MCs in the ultra-low adherent culture dishes (corning). Every two days, 80% percentage of culture medium was aspirated and replaced with an equal volume of fresh medium. To investigate cell attachment and growth, a sample of the MCs was taken at the 1st, 3rd, and 7th day of cell culture and analyzed by fluorescent microscopy after DAPI staining. Accordingly, cells on the surface of the MCs were fixed with paraformaldehyde 4% (Sigma Aldrich) and stained with DAPI (Invitrogen™). Images were taken with a fluorescent microscope (Olympus DP80).
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