Time-lapse imaging assay
-
Cells were transfected with fucci2;mAG-hGem(1/110), which labels nuclei in the S/G2/M phases 2/M marker were grown in 96-well dishes in serum-free DMEM HAM/F12 supplemented with 1×B27 , 20 ng/mL epidermal growth factor (EGF), and 20 ng/mL fibroblast growth factor-2 (FGF-2), at 37°C with 5% CO2.
- The cells were imaged for 96 hours in an incubator on the microscope stage.
- Fifty-one images in the z-axis, both bright-field and single-color (green), were captured at 20-min intervals.
- An inverted microscope (IX-71, Olympus) was fitted with a Nipkow disc scanning confocal unit (CSU10, Yokogawa Electric Corp.), EM-CCD camera (iXON BV-887, Andor), filter wheel, and z motor (Mac5000, Ludl Electronic Products).
- As our imaging device has an attached auto xy stage , several spheres can be monitored in one assay.
- Device control and image analysis were performed using MetaMorph software .
|
Cell culture
-
NSCs isolated from the hippocampi of 6-week-old female Fisher 344 rats , were cultured as previously described on poly-ornithine/laminin-coated plates in medium'>DMEM/F12 medium containing N2 supplement and 20 ng/mL FGF-2 , with subculturing upon reaching 80% confluency using Accutase ( Flow ).
- To induce differentiation, NSCs were cultured for five days in DMEM/F12/N2 medium supplemented with 2% fetal bovine serum (FBS ) and 1 µM retinoic acid (BIOMOL).
- Rat hippocampal astrocytes were isolated from Fisher 344 rats as previously described and cultured on poly-ornithine/laminin-coated plates in DMEM/F12/N2 supplemented with 10% FBS, with subculture upon reaching 90% confluency using Trypsin EDTA .
|
-
Oligodendrocyte. Cells were seeded at a concentration of 5,000 cells/cm2 on tissue culture plastic plates and coverslips and cultured in high glucose DMEM supplemented with 1% Penicillin/Streptomycin , 2 mmol/l L-Glutamine , 1X N1 supplement , 1 µg/ml biotin , 5 ng/ml bFGF , 1 ng/ml PDGF , and 30% B104-conditioned media for 1 day.
- On the second day, CG4 rat oligodendrocyte progenitor cells were added in a co-culture setting to promote differentiation, using co-culture membrane inserts, and the media were changed every 2 days.
- The cells were allowed to differentiate for 5 days and were then fixed and stored in PBS for immunofluorescence.
- Cells were subsequently assessed for expression of the oligodendrocyte markers O2 and NG2.
|
Cell isolation
-
Cells were isolated from the blood vessels in rat, mouse and human.
- Human carotid arteries were obtained from .
- The cells isolation methods were described previously .
- Briefly, the tissue segments were washed three times with phosphate buffered saline (PBS) supplemented with 1% penicillin/streptomycin (P/S).
- The surrounding connective tissues and adventitia were dissected away under a dissecting microscope.
- Endothelium was removed by scraping off the cell layer on the luminal surface with sterile scalpel blades.
- For tissue explant culture method, the tunica media was cut into mm-size and placed onto the surface coated with 1% CellStart in 6-well plates.
- The cells were cultured in MEM with 10% FBS , or in MEM with 2% CEE (MP , ), 1% FBS, 1% N2 , 2% B27 , 100 nM retinoic acid (A) , 50 nM 2-mercaptoethanol (2ME) , 1% P/S and 20 ng/ml bFGF .
- For enzymatic digestion methods, tissues were…
-
Cells were isolated from the blood vessels in rat, mouse and human.
- Human carotid arteries were obtained from .
- The cells isolation methods were described previously .
- Briefly, the tissue segments were washed three times with phosphate buffered saline (PBS) supplemented with 1% penicillin/streptomycin (P/S).
- The surrounding connective tissues and adventitia were dissected away under a dissecting microscope.
- Endothelium was removed by scraping off the cell layer on the luminal surface with sterile scalpel blades.
- For tissue explant culture method, the tunica media was cut into mm-size and placed onto the surface coated with 1% CellStart in 6-well plates.
- The cells were cultured in MEM with 10% FBS , or in MEM with 2% CEE (MP , ), 1% FBS, 1% N2 , 2% B27 , 100 nM retinoic acid (A) , 50 nM 2-mercaptoethanol (2ME) , 1% P/S and 20 ng/ml bFGF .
- For enzymatic digestion methods, tissues were incubated with 3 mg/ml type II collagenase in DMEM with a 1/5 (w/v) ratio of tissue (g) to enzyme solution (ml).
- After incubation at 37°C for 30 min, the same volume of 1 mg/ml elastase solution was added to the solution containing the tissue and collagenase.
- The tissues were incubated for another 1–2 hours until all the tissues were digested.
- Cells were then seeded onto CellStart-coated dishes and maintained at 37°C in an incubator with 5% CO2.
|
Isolation of Skin Derived Progenitor Cells
-
SKPs were isolated from 6 weeks old female Sprague Dawley rats as described previously 2 inhalation.
- Back skin was depilated using wax strips and a piece of skin was removed.
- Skin was washed in cold HBSS and fat layers were removed from the skin underside using forceps.
- Skin was then cut into small pieces using razor blades and digested with collagenase XI (1 mg/ml) /HBSS for 1 hr at 37°C.
- Collagenase/skin cell suspension was passed through a 70 µm mesh to remove remaining tissue pieces.
- Collagenase/cell suspension was diluted with DMEM+Glutamax and cells were isolated by centrifugation (5 min, 1200 rpm).
- Cells were plated in DMEM/F12 3∶1+ Glutamax medium containing 2% B27 , 20 ng/ml EGF , 40 ng/ml FGF2 , Penicillin/Streptomycin and Fungizone .
- Under these conditions, SKPs grow in suspension as spheres.
- For passaging, SKPs spheres were isolated from the culture medium by centrifugation (5 min, 1200 rpm)…
-
SKPs were isolated from 6 weeks old female Sprague Dawley rats as described previously 2 inhalation.
- Back skin was depilated using wax strips and a piece of skin was removed.
- Skin was washed in cold HBSS and fat layers were removed from the skin underside using forceps.
- Skin was then cut into small pieces using razor blades and digested with collagenase XI (1 mg/ml) /HBSS for 1 hr at 37°C.
- Collagenase/skin cell suspension was passed through a 70 µm mesh to remove remaining tissue pieces.
- Collagenase/cell suspension was diluted with DMEM+Glutamax and cells were isolated by centrifugation (5 min, 1200 rpm).
- Cells were plated in DMEM/F12 3∶1+ Glutamax medium containing 2% B27 , 20 ng/ml EGF , 40 ng/ml FGF2 , Penicillin/Streptomycin and Fungizone .
- Under these conditions, SKPs grow in suspension as spheres.
- For passaging, SKPs spheres were isolated from the culture medium by centrifugation (5 min, 1200 rpm) and dissociated into single cells by digestion with collagenase XI.
- For all experiments, passage 3 SKPs were used.
|
Isolation of Skin Derived Progenitor Cells
-
SKPs were isolated from 6 weeks old female Sprague Dawley rats as described previously 2 inhalation.
- Back skin was depilated using wax strips and a piece of skin was removed.
- Skin was washed in cold HBSS and fat layers were removed from the skin underside using forceps.
- Skin was then cut into small pieces using razor blades and digested with collagenase XI (1 mg/ml) /HBSS for 1 hr at 37°C.
- Collagenase/skin cell suspension was passed through a 70 µm mesh to remove remaining tissue pieces.
- Collagenase/cell suspension was diluted with DMEM+Glutamax and cells were isolated by centrifugation (5 min, 1200 rpm).
- Cells were plated in DMEM/F12 3∶1+ Glutamax medium containing 2% B27 , 20 ng/ml EGF , 40 ng/ml FGF2 , Penicillin/Streptomycin and Fungizone .
- Under these conditions, SKPs grow in suspension as spheres.
- For passaging, SKPs spheres were isolated from the culture medium by centrifugation (5 min, 1200 rpm)…
-
SKPs were isolated from 6 weeks old female Sprague Dawley rats as described previously 2 inhalation.
- Back skin was depilated using wax strips and a piece of skin was removed.
- Skin was washed in cold HBSS and fat layers were removed from the skin underside using forceps.
- Skin was then cut into small pieces using razor blades and digested with collagenase XI (1 mg/ml) /HBSS for 1 hr at 37°C.
- Collagenase/skin cell suspension was passed through a 70 µm mesh to remove remaining tissue pieces.
- Collagenase/cell suspension was diluted with DMEM+Glutamax and cells were isolated by centrifugation (5 min, 1200 rpm).
- Cells were plated in DMEM/F12 3∶1+ Glutamax medium containing 2% B27 , 20 ng/ml EGF , 40 ng/ml FGF2 , Penicillin/Streptomycin and Fungizone .
- Under these conditions, SKPs grow in suspension as spheres.
- For passaging, SKPs spheres were isolated from the culture medium by centrifugation (5 min, 1200 rpm) and dissociated into single cells by digestion with collagenase XI.
- For all experiments, passage 3 SKPs were used.
|
Perfusable blood vessel formation and viable cardiac tissue fabrication.
-
Blood vessel formation and tissue viability were evaluated among the four groups, with and without EC coculture, and with or without FGF-2 administration in the perfusion medium.
|
Retrovirus infection and other in vivo manipulations
-
High-titer solutions (2×108 colony forming unit/ml) of recombinant retroviruses pMXIG and its derivatives expressing Neurog2, Pax6, and Ascl1 were prepared with artificial cerebrospinal fluid (124 mM NaCl, 5 mM KCl, 1.3 mM MgCl2, 2 mM CaCl2, 26 mM NaHCO3, and 10 mM D-glucose, with the pH adjusted to 7.2 using aeration with 95% O2–5%CO2) containing rat serum albumin (1 mg/ml).
- In some experiments, the virus solution was supplemented with FGF2 (0.9 μg/ml) and EGF (0.9 μg/ml).
|
Preparation of SLCs
-
After being subcultured at a concentration of 1 × 106 cells/cm2, BM-MSCs were incubated in αMEM containing 1 mM BME without serum for 24 h. The culture medium was then replaced with αMEM containing 10% FBS and 35 ng/ml at-RA .
- After three days, the cells were finally transferred to inducer medium containing αMEM, 10% FBS and trophic factors of 5 μM FSK , 10 ng/ml bFGF , 5 ng/ml PGF , and 200 ng/ml HG .
- The cells were cultured for 10 days [
|
Stability of free versus conjugated neurotrophic factors at 37°C in the absence ((a1), (b1), and (c1)) and in the presence ((a2), (b2), and (c2)) of cells.
-
In the upper row free or conjugated neurotrophic factors (GDNF, βNGF, and FGF-2) were added to culture medium, each type separately (10 ng/mL, final concentration), and placed at 37°C (in the absence of cells).
|
