The O-MCP microchannels were sterilized with ethylene oxide gas and then degassed for 2 days. The microchannels were exposed to oxygen plasma treatment for 1 minute to increase the hydrophilicity of the microchannel surfaces. Microchannels were filled with ethanol, remained filled for 20 minutes, and then rinsed three times with PBS. Microchannels were then filled with a solution of 60 μg/mL collagen from human placenta (Sigma Aldrich, USA) in PBS, and the culture plate was rocked for 1 hour at room temperature. Following three PBS rinses, 15 μL of R-medium was delivered to all microchannel inlet and outlet wells. Medium was aspirated from the top inlet well prior to cell seeding. Thirty microliters of a one million/mL solution of hRPTEC in R-medium was pipetted into the inlet well of each top microchannel. One hour following seeding, R-medium was replaced with 60 μl of fresh medium in all four inlet and outlet wells of each microchannel. Micropump array-driven flow started in all microchannels at 10 μl/min. Two days after seeding, the flow rate increased to 70 μl/min for the remainder of the experiment. R-medium changes occurred every 2 days and 1 hour prior to all oxygen measurements.
Collagen from human placenta
Manufactured by Merck Group
Sourced in United States
Collagen from human placenta is a naturally-derived biomaterial used in various laboratory applications. It is extracted from the human placenta and provides a source of native collagen. The core function of this product is to serve as a substrate or coating material for cell culture, tissue engineering, and other in vitro research purposes.
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Lab products found in correlation
3 protocols using collagen from human placenta
1
Microfluidic Culture of Human Renal Proximal Tubule Cells
2
Intestinal Epithelial Monolayer Differentiation
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Stable
enteroid lines were
grown in matrigel for 5–7 days before breaking the spheres
using a G27 needle and a syringe. The single cell suspension was then
seeded onto well inserts (Corning Transwell clear polyester membrane
0.4 μm, 0.33 cm2, Sigma-Aldrich) coated with collagen
from human placenta (Sigma-Aldrich) and cultured in Intesticult (components
A and B, Stem Cell) for 5–10 days to form confluent monolayers.
The confluency was assessed using a microscope and measuring trans
epithelial resistance (TER) with a Millicell ERS-2 V/ohm meter (Millipore).
When TER reached above 600 Ω/cm2 (minus the well
background resistance), the cells were grown in equal parts Intesticult
component A + DMEM for 5 days to induce the differentiation of the
cells into a more mature state as previously described.42 (link) The differentiated monolayer cultures were then
subjected to an apical CT challenge (0.1 μg/mL) where CT had
been pretreated with polymers or oligosaccharides. To monitor the
CT-induced ion efflux from the TER cells, the trans epithelial resistance
and voltage were monitored, where the short circuit current (Isc) per cm2 was calculated. The results
were then normalized to the measurements on PBS-treated cells.
enteroid lines were
grown in matrigel for 5–7 days before breaking the spheres
using a G27 needle and a syringe. The single cell suspension was then
seeded onto well inserts (Corning Transwell clear polyester membrane
0.4 μm, 0.33 cm2, Sigma-Aldrich) coated with collagen
from human placenta (Sigma-Aldrich) and cultured in Intesticult (components
A and B, Stem Cell) for 5–10 days to form confluent monolayers.
The confluency was assessed using a microscope and measuring trans
epithelial resistance (TER) with a Millicell ERS-2 V/ohm meter (Millipore).
When TER reached above 600 Ω/cm2 (minus the well
background resistance), the cells were grown in equal parts Intesticult
component A + DMEM for 5 days to induce the differentiation of the
cells into a more mature state as previously described.42 (link) The differentiated monolayer cultures were then
subjected to an apical CT challenge (0.1 μg/mL) where CT had
been pretreated with polymers or oligosaccharides. To monitor the
CT-induced ion efflux from the TER cells, the trans epithelial resistance
and voltage were monitored, where the short circuit current (Isc) per cm2 was calculated. The results
were then normalized to the measurements on PBS-treated cells.
3
Intestinal Enteroid Monolayer Infection Assay
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The secretor-positive jejunal J2 human intestinal enteroids were maintained as previously described [23 (link), 34 (link)]. For infections, monolayer cultures in 96-well plates were prepared as described previously [23 (link)]. In short, plates were coated with collagen from human placenta (Sigma-Aldrich). The enteroid cultures were washed with 0.5 mM EDTA in ice-cold phosphate-buffered saline (PBS), without CaCl2 and MgCl2, and dissociated with 0.05% trypsin/0.5 mM EDTA for 4 minutes at 37°C, trypsin was inactivated by adding 10% fetal bovine serum diluted in complete medium without growth factors (CMGF−). Next, cells were passed through a 40-μm cellstrainer and seeded in 96-well plates in complete medium with growth factors (CMGF)+ containing ROCK inhibitor (10 μM, Sigma-Aldrich). After 24 hours, the CMGF+ was replaced with differentiation medium to allow differentiation for 4 days [23 (link)].
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