This method produces highly reproducible hepatocyte-like cells in a 96 well format by using semi-automated platforms. The main advantage of this technique is that it's rapid, cost-effective, with reduced variation and allows the mass production of 96 well plates of hepatocyte-like cells. Prepare a five microgram per milliliter solution of thawed laminin 521 by diluting it in ice-cold one time Dulbecco's phosphate-buffered saline or DPBS with calcium magnesium.
Use an automatic liquid handling dispenser with a standard tube dispensing cassette to add 50 microliters of the laminin 521 solution per well of a 96 well plate. Incubate the laminin 521 coated plates. After incubation, aspirate the laminin 521 solution off the coated surface, using an eight channel aspiration adaptor and avoid any contact with the coated surface.
Immediately add 50 microliters per well of mTeSR1 medium supplemented with 10 micromolar of Rho-associated kinase or ROCK inhibitor Y27632. Keep the plate in incubator until cell seeding at 37 degrees celsius and 5%CO2. From a cell suspension of three times 10 to the fifth cells per milliliter in mTeSR1 medium supplemented with 10 micromolar ROCK inhibitor Y27632.
Add 50 microliters of the cell suspension onto the plate. After seeding, return the plates to the cell incubator at 37 degrees Celsius, 5%CO2, for 24 hours to allow the cells to attach. After taking the plate from the incubator, examine the plate next day and check that cell to cell contact has been established and confluency reaches 40%Then switch to differentiation media.
Cell confluency appeal the starting of the differentiation. It's key to achieve good quality of hepatocyte like cells. The start of the differentiation, once the confluency reaches 40%If the confluency is higher, plates are not suited well for differentiation.
24 hours post seeding, carefully remove the medium using an automatic hand-held electronic channel pipette. And add 100 microliters of fresh endoderm priming medium supplemented with 100 nanograms per milliliter activin A and 50 nanograms per milliliter Wnt3a. After definitive endoderm induction, switch to KSR DMSO medium for hepatoblast specification.
And change the medium every two days for five days. Following the hepatoblast differentiation, remove the KSR DMSO medium and wash the cells once with HepatoZYME medium without the supplement. After the wash, add 100 microliters of HepatoZYME maturation medium supplemented with 10 nanograms per milliliter hepatocyte growth factor and 20 nanograms per milliliter Oncostatin M.Change the medium every 48 hours for seven to ten days.
On day 18 of differentiation, fix the cells and perform immunocytochemistry, using the liquid handling and automatic plate washer. Store the plates at four degrees celsius in the dark until imaging. Follow with imaging and quantification.
On day 18, the variability and cell number between wells was examined following DAPI staining. After seven fields of view were captured per well and the number of nuclei quantified, no statistical differences were identified. Staining of human hepatocyte like cells or HLCs at day 18 showed expression of hepatocyte markers HNF4 alpha, Albumin, and AFP.
HLCs also expressed CYP P450 proteins, CYP2D6, and CYP3A4 showed CYP P450 metabolic activity. HLCs also displayed a distinct polygonal appearance, showed by E-cadherin. Despite the standardization of this protocol, cell confluency prior to differentiation is critical for HRCs differentiation.
We see cell independent. Therefore, sell seeding and the density optimization is required for each cell line. Once mastered, this methodology allows the rapid generation of multiple plates of human liver cells for disease modeling, drug screening, or drug proposing studies.
This systems allows the generation of multiparametrical datasets for mechanistic analysis for hepatocyte biology.
