Two hESC lines (Regea 08/023; 46, XY, Regea 08/017; 46,XX) [50 (link)] and one human induced pluripotent stem cell (hiPSC) line, (UTA.04511.WTS 46, XY) [51 (link)] were used for this study. Cell lines were cultured on top of mitomycin-treated (10 μg/ml,Sigma-Aldrich) (i.e. mitotically inactivated) human foreskin fibroblasts feeder cells (CRL-2429TM, ATCC, Manassas, VA, USA). The undifferentiated cells were cultured similarly as in Sorkio et al. [52 (link)] and after one week of culture the differentiation was induced by reducing the KO-SR concentration to 15%, removing the bFGF and commencing the floating culture as previously described in Vaajasaari et al. [53 (link)]. Floating aggregates were fed thrice a week and grown for 70–195 days. The pigmented areas of floating aggregates were manually dissected, dissociated with 1x Trypsin-EDTA and replated on collagen IV from human placenta (5 μg/cm2, Sigma-Aldrich). Adherently cultured cells were imaged for the fusiform morphology after 8 days (range 6–9 days), for the epithelioid morphology after 9 days (range 8–9) and for the cobblestone morphology after 19 days (range 17–24) of culturing.
Collagen 4 from human placenta
Manufactured by Merck Group
Sourced in United States
Collagen IV from human placenta is a purified extracellular matrix protein. It is a structural component of basement membranes.
Automatically generated - may contain errors
Lab products found in correlation
Ham s f12, by Thermo Fisher Scientific (5 mentions)
Human endothelial sfm, by Thermo Fisher Scientific (5 mentions)
Dulbecco s phosphate buffered saline, by Thermo Fisher Scientific (5 mentions)
Medium 199, by Thermo Fisher Scientific (5 mentions)
Liberase th, by Roche (5 mentions)
Gentamicin, by Thermo Fisher Scientific (4 mentions)
Penicillin, by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Amphotericin b, by Thermo Fisher Scientific (4 mentions)
Equafetal, by Atlas Biologicals (4 mentions)
Y27632, by Miltenyi Biotec (3 mentions)
Tryple select, by Thermo Fisher Scientific (3 mentions)
Ascorbic acid, by Avantor (3 mentions)
Insulin transferrin selenium, by Thermo Fisher Scientific (2 mentions)
Ascorbic acid, by Merck Group (2 mentions)
Trypan blue, by Merck Group (2 mentions)
Insulin transferrin selenium, by Corning (2 mentions)
Crl 2429, by American Type Culture Collection (1 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (1 mentions)
Recombinant basic fibroblast growth factor bfgf, by R&D Systems (1 mentions)
10 protocols using collagen 4 from human placenta
1
Differentiation of hESC and hiPSC into RPE Cells
2
Isolation and Characterization of Human Endothelial Cells
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Ham’s F12, Medium 199, Human Endothelial-SFM, Minimum Essential Medium (MEM), Dulbecco’s Phosphate-Buffered Saline (PBS), Insulin/Transferrin/Selenium (ITS), TrypLE Express (TE), 1 M HEPES buffer, and antibiotics, were all purchased from Life Technologies (Carlsbad, CA, USA). Trypan blue (0.4%), alizarin red, paraformaldehyde (PFA), Triton X-100, Collagen IV from human placenta, and ascorbic acid were purchased from Sigma (St. Louis, MO, USA). Calcein AM, propidium iodide (PI), Hoechst 33342, and PureLink RNA Micro kit were obtained from Invitrogen (Carlsbad, CA, USA). Recombinant basic fibroblast growth factor (bFGF, R&D, Minneapolis, MN, USA). Rho-associated, coiled-coil protein kinase (ROCK) inhibitor, Y-27632 (Miltenyi Biotec, Bergisch Gladbach, Germany). FNC coating mixture (United States Biologicals, Swampscott, MA, USA). Liberase TH (Roche Mannhein, Germany). EquaFetal serum was obtained from Atlas Biologicals (Fort Collins, CO, USA). Optisol-GS was purchased from Basuch & Lomb (Rochester, NY, USA).
3
Cryosectioning and FTIR Spectroscopy
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The reference alginate hydrogel sample was prepared by embedding, freezing and sectioning; cryosections 16 μm thick were placed on a CaF2 slide. The reference spectrum of collagen was acquired from collagen IV from human placenta (Sigma−Aldrich, Steinheim, Germany). The reference spectrum of sucrose was acquired from sucrose solution dried on a CaF2 slide. Reference spectra were collected in transmission mode with a single-channel MCT detector using the same acquisition parameters as for imaging of spinal cord samples.
4
Culturing CuFi-1 and NuLi-1 Cell Lines
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The CuFi-1 and NuLi-1 cell lines were a generous gift of A. Klingelhuts, Pkarp, and J. Zbaner, University of Iowa, Iowa City [27 (link)], and were cultured as previously described [25 (link)]. Briefly, the cells were cultured as monolayer in a humidified atmosphere at 37°C and 5% CO2 in flasks precoated with collagen (collagen IV from human placenta, Sigma-Aldrich) in bronchial epithelial growth medium (BEGM by Lonza; singleQuot Kit Lonza).
5
SH-SY5Y Neuronal Differentiation Protocol
6
Isolation and Culture of Human Endothelial Cells
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Ham’s F12, Medium 199, Human Endothelial-SFM, fetal bovine serum (FBS), Dulbecco’s Phosphate-Buffered Saline (PBS), Insulin/Transferrin/Selenium (ITS), Collagenase Type I, TrypLETM Express (TE), TrypLETM Select (TS), gentamicin, amphotericin B, 5-ethynyl-29-deoxyuridine (EdU) incorporation Click-iT Alexa Fluor 488 cell proliferation assay kit, Fix and Perm (Medium A), penicillin and streptomycin were purchased from Life Technologies (Carlsbad, CA, USA). Dimethyl sulfoxide (DMSO), trypan blue (0.4%), alizarin red, paraformaldehyde (PFA), human serum (HS), Collagen IV from human placenta, ascorbic acid, and ascorbate-2-phosphate were purchased from Sigma (St. Louis, MO, USA). Recombinant basic fibroblast growth factor was bought from R&D Systems (bFGF, Minneapolis, MN, USA). Rho-associated, coiled-coil protein kinase inhibitor Y-27632 was brought from Miltenyi Biotec (Bergisch Gladbach, Germany). FNC coating mixture was obtained from United States Biologicals (Swampscott, MA, USA). Liberase TH was purchased from Roche (Mannhein, Germany). EquaFetal® was from Atlas Biologicals (Fort Collins, CO, USA). FREEZEstem cryo-preservation medium, LAMSCREENTM, human recombinant laminin-511 and 521 were bought from BioLamina (Sundbyberg, Sweden). Finally, Cryoscarless cryo-preservation medium was from Funakoshi (Tokyo, Japan).
7
Culturing Rat Pheochromocytoma (PC12) Cells
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Rat adrenal pheochromocytoma 12 (PC12) cells in 10% DMSO (Greene & Tischler, 1976 (link)) were thawed from liquid nitrogen storage in a 37°C water bath, centrifuged, and resuspended in RPMI‐1640 medium (Sigma‐Aldrich) supplemented with 1% (v/v) penicillin–streptomycin (Invitrogen), 1% (v/v) l ‐glutamine (200 mM; Invitrogen), and 10% (v/v) horse serum (Invitrogen), mechanically dissociated, and plated on 25cm2 cell culture flasks (Fisher Scientific) previously coated for 1 h with 1 mg/ml Collagen IV from human placenta (Sigma) dissolved in acetic acid and diluted 10× in 0.01% w/v Poly‐L‐Lysine solution (Sigma), followed by washing with PBS (Sigma). When grown to 90% confluency, PC12 cells were detached from the flasks using 0.05% trypsin (Thermo Fisher Scientific), centrifuged, resuspended in RPMI, and mechanically dissociated. Cells were then seeded on pre‐coated coverslips. Cells were kept in an incubator at 37°C and 5% CO2 for 18–36 h before transfection, by which time they had attained 50–80% confluency.
8
Isolation and Culture of Human Endothelial Cells
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Ham’s F12, Medium 199, Human Endothelial-SFM, Dulbecco’s Phosphate-Buffered Saline (PBS), TrypLETM Select (TS), gentamicin, amphotericin B, penicillin and streptomycin were purchased from Life Technologies (Carlsbad, CA, USA). Insulin/Transferrin/Selenium (ITS) was purchased from Corning (New York, NY, USA), and ascorbic acid from Avantor (Pennsylvania USA). Collagen IV from human placenta and Trypan blue (0.4%) were purchased from Sigma (St. Louis, MO, USA). Recombinant human basic fibroblast growth factor and rho-associated, coiled-coil protein kinase inhibitor Y-27632 were brought from Miltenyi Biotec (Bergisch Gladbach, Germany). The FNC coating mixture was obtained from United States Biologicals (Swampscott, MA, USA). Liberase TH was purchased from Roche (Mannhein, Germany). EquaFetal® was from Atlas Biologicals (Fort Collins, CO, USA).
9
Endothelial Cell Culture Protocol
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Ham’s F12, Medium 199, Human Endothelial-SFM, Dulbecco’s Phosphate-Buffered Saline (PBS), TrypLETM Select (TS), gentamicin, amphotericin B, penicillin and streptomycin were purchased from Life Technologies (California, USA). Dimethyl sulfoxide (DMSO), trypan blue (0.4%), alizarin red, paraformaldehyde (PFA), and Collagen IV from human placenta were purchased from Sigma (Missouri, USA). Human recombinant basic fibroblast growth factor (HrFGF), and Rho-associated, coiled-coil protein kinase inhibitor (ROCKi), Y-27632, was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Liberase TH was purchased from Roche (Mannhein, Germany). EquaFetal® was from Atlas Biologicals (Colorado, USA). Insulin/Transferrin/Selenium (ITS) was purchased from Corning (New York, USA), and ascorbic acid from Avantor (Pennsylvania USA).
10
Endothelial Cell Culture Protocol
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Ham’s F12, Medium 199, Human Endothelial-SFM, Dulbecco’s Phosphate-Buffered Saline (PBS), TrypLETM Select (TS), gentamicin, amphotericin B, penicillin, and streptomycin were purchased from Life Technologies (Carlsbad, CA, USA). Insulin/Transferrin/Selenium (ITS) was purchased from Corning (Corning, NY, USA), and ascorbic acid was purchased from Avantor (Radnor Township, PA, USA). Collagen IV from human placenta and Trypan blue (0.4%) were purchased from Sigma (St. Louis, MO, USA). Recombinant human basic fibroblast growth factor (bFGF) and rho-associated, coiled-coil protein kinase inhibitor Y-27632 were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). FNC coating mixture was obtained from United States Biologicals (Swampscott, MA, USA). Liberase TH was purchased from Roche (Mannhein, Germany). EquaFetal®, the bovine serum used to supplement the culture medium, was from Atlas Biologicals (Fort Collins, CO, USA).
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