urine osmolarity  (Advanced Instruments Inc)

Journal: JOR Spine

Article Title: Hyperosmolar expansion medium improves nucleus pulposus cell phenotype

doi: 10.1002/jsp2.1219

Figure Lengend Snippet: Expansion in 500 mOsm/L results in polygonal nucleus pulposus cells (NPCs) with a lower cell proliferation rate. (A) Experimental design. (B) Population doubling time of the NPCs during Passage 1 to 2 and 2 to 3 when expanded in normoxia (NX: 21% O 2 ) and hypoxia (HX: 5% O 2 ) in the different media osmolarities (lines indicate the mean ± SD). The black symbols represent female donors and white symbols male donors. * p < 0.05 corrected for multiple testing. (C) Representative brightfield images of NPC morphology at Day 3 of expansion in both NX and HX conditions in the different media osmolarities

Article Snippet: Cryopreserved P0 NPCs were expanded in medium containing hgDMEM+Glutamax (31966, Invitrogen) with 10% fetal bovine serum (FBS) (16000–044, Life Technologies), 1% penicillin/streptomycin (P/S, P11‐010, PAA Laboratories), 0.1 mM ascorbic acid 2‐phosphate (Asap, A8960, Sigma‐Aldrich), and 1 ng/ml bFGF (PHP105, AbD Serotec) at standard osmolarity (300 mOsm/L ) or adjusted to 400 using 1% 5 M NaCl (6404; Merck) and 1% 0.4 M KCl (P4505; Sigma‐Aldrich) or to 500 mOsm/L using 2% 5 M NaCl and 2% 0.4 M KCl.

Techniques:

Journal: JOR Spine

Article Title: Hyperosmolar expansion medium improves nucleus pulposus cell phenotype

doi: 10.1002/jsp2.1219

Figure Lengend Snippet: Hyperosmolar expansion medium improves the phenotype of dog nucleus pulposus cells (NPCs) independent of oxygen level. (A) Fold changes in relative gene expression of NPC and progenitor markers after expansion of two passages in both normoxic (NX: 21% O 2 ) and hypoxic (HX: 5% O 2 ) conditions in the different media osmolarities (lines indicate the mean + SD). The black symbols represent female donors and white symbols represent male donors. * p < 0.05 corrected for multiple testing. (B) Immunofluorescence images and quantification for ACAN, PAX1, CD24, TEK, and CD73 protein levels following expansion for two passages in the different media osmolarities in HX (lines indicate the mean ± SD). The black symbols represent female donors and white symbols represent male donors. ** p < 0.01, *** p < 0.001, #significantly different ( p < 0.05) from expansion in HX using the same medium osmolarity corrected for multiple testing

Article Snippet: Cryopreserved P0 NPCs were expanded in medium containing hgDMEM+Glutamax (31966, Invitrogen) with 10% fetal bovine serum (FBS) (16000–044, Life Technologies), 1% penicillin/streptomycin (P/S, P11‐010, PAA Laboratories), 0.1 mM ascorbic acid 2‐phosphate (Asap, A8960, Sigma‐Aldrich), and 1 ng/ml bFGF (PHP105, AbD Serotec) at standard osmolarity (300 mOsm/L ) or adjusted to 400 using 1% 5 M NaCl (6404; Merck) and 1% 0.4 M KCl (P4505; Sigma‐Aldrich) or to 500 mOsm/L using 2% 5 M NaCl and 2% 0.4 M KCl.

Techniques: Gene Expression, Immunofluorescence

Journal: JOR Spine

Article Title: Hyperosmolar expansion medium improves nucleus pulposus cell phenotype

doi: 10.1002/jsp2.1219

Figure Lengend Snippet: Hyperosmolar expansion medium maintains healthy ECM production and deposition during 3D culture. (A) GAG and DNA content of the nucleus pulposus cell (NPC) microaggregates at Day 14 of 3D culture (300 mOsm/L; 5% O 2 ) after expansion in both normoxic (NX; 21% O 2 ) and hypoxic (HX; 5% O 2 ) conditions in the different media osmolarities (300, 400, and 500 mOsm/L; lines indicate the mean ± SD). The black symbols represent female donors and white symbols represent male donors (mean of two technical replicates). * p < 0.05, ** p < 0.01, *** p < 0.001 corrected for multiple testing. (B) Safranin O/Fast Green and immunohistochemical staining for aggrecan and collagen type II of the NPC microaggregates collected at Day 14 of 3D culture (300 mOsm/L; 5% O 2 ) after expansion in HX conditions in the different media osmolarities

Article Snippet: Cryopreserved P0 NPCs were expanded in medium containing hgDMEM+Glutamax (31966, Invitrogen) with 10% fetal bovine serum (FBS) (16000–044, Life Technologies), 1% penicillin/streptomycin (P/S, P11‐010, PAA Laboratories), 0.1 mM ascorbic acid 2‐phosphate (Asap, A8960, Sigma‐Aldrich), and 1 ng/ml bFGF (PHP105, AbD Serotec) at standard osmolarity (300 mOsm/L ) or adjusted to 400 using 1% 5 M NaCl (6404; Merck) and 1% 0.4 M KCl (P4505; Sigma‐Aldrich) or to 500 mOsm/L using 2% 5 M NaCl and 2% 0.4 M KCl.

Techniques: Immunohistochemical staining, Staining

Journal: JOR Spine

Article Title: Hyperosmolar expansion medium improves nucleus pulposus cell phenotype

doi: 10.1002/jsp2.1219

Figure Lengend Snippet: Nucleus pulposus cells (NPCs) expanded in higher osmolarity retain the expression of healthy phenotypic markers during 3D culture. Immunopositivity for PAX1, CD24, TEK, and CD73 at Day 14 in NPC microaggregates collected at Day 14 of 3D culture (300 mOsm/L; 5% O 2 ) after expansion in hypoxic conditions in the different media osmolarities. Arrows indicate extracellular matrix, cytoplasmic/membranous and arrow heads indicate nuclear localization of immunopositivity.

Article Snippet: Cryopreserved P0 NPCs were expanded in medium containing hgDMEM+Glutamax (31966, Invitrogen) with 10% fetal bovine serum (FBS) (16000–044, Life Technologies), 1% penicillin/streptomycin (P/S, P11‐010, PAA Laboratories), 0.1 mM ascorbic acid 2‐phosphate (Asap, A8960, Sigma‐Aldrich), and 1 ng/ml bFGF (PHP105, AbD Serotec) at standard osmolarity (300 mOsm/L ) or adjusted to 400 using 1% 5 M NaCl (6404; Merck) and 1% 0.4 M KCl (P4505; Sigma‐Aldrich) or to 500 mOsm/L using 2% 5 M NaCl and 2% 0.4 M KCl.

Techniques: Expressing