Approximately 200,000 DCs were placed in a polypropylene conical tube (Corning Inc., USA) with 0.5 ml of chondrogenic media (DMEM-high glucose added with insulin, transferrin, and selenium (ITS-G; Thermo Fisher Scientific, MA, USA)), 5 mg/ml linoleic acid, 10 ng/ml transforming growth factor beta (TGF-β1; R&D Systems, MN, USA), 14 mg/ml ascorbic acid, 10− 7 M dexamethasone (Sigma-Aldrich, MS, USA), 1.0 mg/ml human serum albumin (Equitech-Bio Inc., KV, USA), and 1% penicillin/streptomycin). The cells were centrifuged (470×g at 4 °C for 5 min) and incubated (37 °C and 5% CO2) for 3–4 h to allow spheroid formation. For the EV treatment group, the media were replaced with 500 μl of chondrogenic media containing sEVs (5 × 1010 vesicles/ml). Chondrogenic media without the sEVs served as control. The media were replaced with fresh media with or without sEVs every 48 h, the used media were collected and centrifuged (300×g, 4 °C, 5 min), and the supernatants were stored at − 80 °C for further analysis. The pellets were then harvested at days 7, 14, and 28. Four replicates of pellets were cultured for each group and repeated with different donor cells (n = 5) separately. Two replicates were used for histology and two for biochemical analysis.
Human serum albumin (hsa)
Manufactured by Equitech-Bio
Sourced in United States
Human serum albumin is a protein found in human blood plasma. It serves as the main transport protein in the circulatory system, responsible for maintaining the osmotic pressure and pH balance of the blood.
Automatically generated - may contain errors
Lab products found in correlation
Dexamethasone (dex), by Merck Group (5 mentions)
Tgf β1, by R&D Systems (3 mentions)
Its g, by Thermo Fisher Scientific (2 mentions)
Ascorbic acid, by Merck Group (2 mentions)
Insulin, by Thermo Fisher Scientific (2 mentions)
Linoleic acid solution, by Merck Group (2 mentions)
Its g premix, by Thermo Fisher Scientific (2 mentions)
Polypropylene conical tube, by Corning (1 mentions)
Penicillin streptomycin pest, by Merck Group (1 mentions)
Insulin transferrin selenium, by Thermo Fisher Scientific (1 mentions)
Dmem hg, by Thermo Fisher Scientific (1 mentions)
Linoleic acid, by Merck Group (1 mentions)
Mmp 1, by Thermo Fisher Scientific (1 mentions)
Mmp 1, by R&D Systems (1 mentions)
Transferrin, by Thermo Fisher Scientific (1 mentions)
Bmp 2, by Merck Group (1 mentions)
L ascorbic acid, by Merck Group (1 mentions)
Selenous acid, by Thermo Fisher Scientific (1 mentions)
Tgf β3, by Merck Group (1 mentions)
Via1 casettes, by ChemoMetec (1 mentions)
5 protocols using human serum albumin (hsa)
1
Chondrogenic Differentiation of DCs with sEVs
2
Chondrocyte Expansion and Differentiation
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Chondrocytes was expanded in monolayer as earlier described [42] (link). Subsequently passage 3 cells were seeded at 20, 000 cells/cm2 in Dulbecco’s modified Eagles medium-high glucose (DMEM-HG) (Thermo Fisher Scientific; Waltham, MA, USA) supplemented with 14 μg/ml ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA), 10−7 M dexamethasone (Sigma-Aldrich), 1 mg/mL human serum albumin (Equitech Bio, Kerville, TX, USA), 1 × insulin–transferrin–selenium (Gibco, Life Technologies, Carlsbad, CA, USA), 5 μg/mL linoleic acid (Sigma-Aldrich), 1 × penicillin-streptomycin (PEST) (Sigma-Aldrich), and 10 ng/mL human transforming growth factor (TGF) β-1 (R&D Systems, Abingdon, UK) on glass coverslips (Nr 1, diameter 20 mm, BergmanLabora, Stockholm, Sweden) in 12 well culture plates for Ca2+ and immunohistochemistry analysis, or in 6 well culture plates for Western blot analysis.
3
Chondrocyte Response to MMP-1 Exposure
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Three stimulation groups were performed on DC pellets, namely the MMP-1, the CM + MMP-1, and the control group. Pellets in the MMP-1 group were cultured in chondrogenic media: DMEM-high glucose added with insulin, transferrin, and selenium (ITS-G; Thermo Fisher Scientific), 5 mg/mL linoleic acid, 10 ng/mL transforming growth factor-β (TGF-β1; R&D Systems, MN, USA), 14 mg/mL ascorbic acid , 10 -7 M dexamethasone (Sigma-Aldrich, MO, USA), 1.0 mg/mL human serum albumin (Equitech-Bio Inc., Kerrville, TX, USA), and 1% penicillin/streptomycin). Based on the concentration of MMP-1 detected in disk tissue from patients diagnosed with degenerative disk disease, concentrations of 5, 50, or 100 ng/mL MMP-1 (R&D Systems, Minneapolis, MN, USA) were selected and added to the pellet from days 14-28 during every media change. MMP-1 was added on day 14 in order to allow the cells to start producing matrix within the pellets before the matrix-degrading enzyme was added. Pellets in the CM + MMP-1 group were cultured in CM supplied with chondrogenic media in a 1: 1 ratio to replenish the nutrients depleted. MMP-1 at concentrations of 5, 50, or 100 ng/mL were added on days 14-28. Pellets treated with chondrogenic media alone without addition of MMP-1 or CM served as controls.
4
iPSC-Chondrocyte Constructs for Cartilage Regeneration
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Equal numbers of iPSCs were mixed with iChons to give a final concentration of 20 million cells/ml in the bioink, and the printed constructs were maintained at 37 °C and 90% humidity in 5% CO2. After 7 days of culture in the DEF CS™ (TaKaRa ClonTech, Sweden) pluripotent medium mixed with an equal volume of conditioned DEF-CS medium (conditioned DEF medium was taken from 80% confluent pluripotent iPSCs after 24 hours of culture, sterile filtered, and fresh growth factors (GF1, GF2 and GF3; TaKaRa ClonTech, Sweden) were added), the medium was changed every day. Cells were then initiated to differentiate by changing the medium to a defined chondrogenic medium (high-glucose Dulbecco’s modified Eagle’s medium; PAA Laboratories) supplemented with 5.0 μg/ml linoleic acid solution (Sigma-Aldrich), 1x ITS-G premix (10 mg/l insulin, 5.5 mg/l transferrin, 6.7 μg/l selenious acid; Life Technologies), 0.11g/l sodium pyruvate, 1.0 mg/ml human serum albumin (Equitech-Bio, TX, USA), 10 ng/ml TGFβ1 (R&D Systems, Abingdon, UK), 10 ng/ml GDF5, 10 ng/ml BMP2, 100 nM dexamethasone (Sigma-Aldrich), 80 μM L-ascorbic acid (Sigma-Aldrich), and 1x penicillin/streptomycin (PEST; PAA Laboratories). The medium was changed three times a week. Relevant control cultures with only printed irradiated chondrocytes were kept throughout the differentiation protocol.
5
Chondrocyte Microtissue Formation and Characterization
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Control chondrocytes (200,000/well) were centrifuged to form pellets in a 96-well
round-bottom low-attachment plate. The plate was centrifuged for 5 minutes at
500 × g to form pellets, with one in each 96 well to form a
micro tissue following differentiation for at least 2 weeks. Then, the 3D
bioprint with and without chondrocytes and the control chondrocyte micro tissue
pellets were induced to differentiate by replacing the defined medium (DEF) with
chondrogenic differentiation medium, consisting of DMEM-high-glucose
(high-glucose DMEM; PAA Laboratories) supplemented with 5.0 mg/mL linoleic acid
solution (Sigma-Aldrich), 1× ITS-G premix (6.25 mg/mL insulin, 6.25 mg/mL
transferrin, 6.25 ng/mL selenous acid; Life Technologies), 1.0 mg/mL human serum
albumin (Equitech-Bio, Kerrville, TX, USA), 10 ng/mL TGFβ1, 10 ng/mL TGFβ3, 100
nM dexamethasone (Sigma-Aldrich), 80 nM ascorbic acid 2 phosphate
(Sigma-Aldrich), and 1× penicillin/streptomycin (PEST; PAA Laboratories). The
medium was changed 3 times a week. The 3D bioprints and control chondrogenic
pellets were harvested after 14 days for histological and reverse
transcription–polymerase chain reaction (RT-PCR) analysis. The cell number and
viability were counted before and after 3D bioprinting using nucleocounter
NC-200TM in Via1-CasettesTM (ChemoMetec, Denmark).
round-bottom low-attachment plate. The plate was centrifuged for 5 minutes at
500 × g to form pellets, with one in each 96 well to form a
micro tissue following differentiation for at least 2 weeks. Then, the 3D
bioprint with and without chondrocytes and the control chondrocyte micro tissue
pellets were induced to differentiate by replacing the defined medium (DEF) with
chondrogenic differentiation medium, consisting of DMEM-high-glucose
(high-glucose DMEM; PAA Laboratories) supplemented with 5.0 mg/mL linoleic acid
solution (Sigma-Aldrich), 1× ITS-G premix (6.25 mg/mL insulin, 6.25 mg/mL
transferrin, 6.25 ng/mL selenous acid; Life Technologies), 1.0 mg/mL human serum
albumin (Equitech-Bio, Kerrville, TX, USA), 10 ng/mL TGFβ1, 10 ng/mL TGFβ3, 100
nM dexamethasone (Sigma-Aldrich), 80 nM ascorbic acid 2 phosphate
(Sigma-Aldrich), and 1× penicillin/streptomycin (PEST; PAA Laboratories). The
medium was changed 3 times a week. The 3D bioprints and control chondrogenic
pellets were harvested after 14 days for histological and reverse
transcription–polymerase chain reaction (RT-PCR) analysis. The cell number and
viability were counted before and after 3D bioprinting using nucleocounter
NC-200TM in Via1-CasettesTM (ChemoMetec, Denmark).
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