Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces

The overall goal of this procedure is to optimize polymer coated surfaces to preserve the long-term stable culture of stem cell derived human hepatocytes. This is accomplished by first synthesizing polyurethane 1 3 4 or PU 1 3 4. The second step is to choose the right solvent to solubilize PU 1 34.

Next, the spin coating process with the PU 1 3 4 on the glass cover slip is optimized. The final step is optimization of the sterilization procedure of the polymer P 1 34 coated glass cover slips ultimately scanning electron microscopy, atomic force microscopy and drug metabolism. Assays are used to show the influence of the PU 1 34 polymer coating in the function of stem cell derived hepatocytes.

The main advantage of this technique over existing methods like the use of al Metrices in culture in maintaining URI potent stem cells that have hepatocytes, is that these synthetic materials can be fine tuned for purpose and a s scale to quality assured standards. In addition, they display batch two bus consistency. The implications of this technique extend toward therapy of liver diseases because it represents an example of how the surface of a synthetic polymer can be optimized to preserve cell phenotype.

Demonstrating the procedure will be Dr.Jeffrey Walton, a postdoc from my laboratory Prior to starting this procedure. Apply heat treatment to the mono’s one six hexane dial di ethylene glycol and neo PenTile glycol at 40 degrees Celsius for 48 hours in a vacuum oven. To remove any residual water, add 22 millimoles of each monomer and 55 millimoles of a dpic acid to a two necked round bottom flask connected to a dean stark apparatus and equipped with a magnetic stir bar.

Following this place the whole assembly under a vacuum and gently heat the glassware at 40 degrees Celsius for six hours in order to avoid any moisture absorption during the addition of the chemical to the flask. After removing the assembly from the oven and placing the system under nitrogen, add 0.0055 moles of the catalyst titanium four, but oxide dropwise by a syringe to the flask. Stir the reaction mixture at 180 degrees Celsius for 24 hours, collecting residual water in the dean stark trap.

Allow the product to cool to room temperature. Next mix 3.2 millimole, or one equivalent of the polyol P-H-N-G-A-G with 6.4 milli mole or two equivalents of four four methyl and a bisphenol isocyanate. In 12 milliliters of anhydrous DML to a two necked round bottom flask connected to a dean stark apparatus and equipped with a magnetic star bar.

Stir the reaction mixture at 70 degrees Celsius under a nitrogen atmosphere. Then add the catalyst titanium four oxide dropwise via syringe to a final concentration of 0.8%After one hour, add one equivalent of the chain extender one four butane dial to give copolymer product. Increase the temperature to 90 degrees Celsius and starve for 24 hours under a nitrogen atmosphere.

Once the reaction mixture has called to room temperature a dal ether dropwise to the reaction mixture until precipitation of the polyurethane product is observed. After transferring the reaction mixture to a centrifuge tube centrifuge the solution at 5, 300 times G for five minutes. After decanting, the supinate dry off at room temperature until the solvent evaporates.

Operates next weigh 200 milligrams of PU 1 34 into a glass bottle. Dilute the sample to a final concentration of 2%in chloroform or chloroform and toluene in a one-to-one ratio or tetra roran or a combination of tetra roran anti chloro methane. In a one-to-one ratio shake the solution vigorously for 20 minutes at room temperature using a shaker at a speed of 200 motions per minute until the solution becomes homogeneous and no precipitate is observed.

When finished, place around 15 millimeter square glass cover slip on the spin coter. Apply 50 microliters of the PU one through four solution to the cover slip using a pipette spin each cover slip for seven seconds at 23 times G.Then air dry the cover slip at room temperature for at least 24 hours before sterilization. At this point, gamma irradiate each polymer coated cover slip by applying a dose of 10 grays using a laboratory radiator for 12 minutes.

Alternatively, UV irradiate each polymer coated cover slip using a 30 watt UV bulb for 16 minutes on each side. When finished, place the polymer coated cover slip in a suitable tissue culture plate according to the cover slip size. After culturing and differentiating the human embryonic stem cell line detached cells using dissociation reagent and replay them onto the PU 1 34 coated cover slips at day nine of the differentiation process in the presence of serum free medium scanning electron microscopy.

Images of the different coated glass cover slips show that the coating with toluene or chloroform was not homogenous, demonstrating an uneven coating with PE 1 34 precipitation. In contrast, tetrahedran allowed the spin coating of a much more uniform surface. Additionally, atomic force microscopy analysis of the different polymer coatings shows a 40%reduction in the roughness of P 1 34 solubilized in tetra hydro furin in comparison to other solvents demonstrating a more uniform PU 1 34 coating a biological application hepatic endoderm differentiation was induced and at day nine, hepato blasts like cells were apparent with the majority of cells expressing cytokeratin 19 alpha feta protein and hepatocyte nuclear factor for alpha and low levels of albumin as a measure of metabolic function.

Cytochrome P four 50 function was assessed four days post replating onto different polymer coated surfaces. A twofold increase in metabolic activity in cells replated on tetra hydro fure mpu 1 3 4 coated surfaces was observed. These results demonstrate that human embryonic stem cell derived hepatocyte metabolic activity is improved with a more uniform P 1 34 coating face Contrast imaging showed no gross differences post UV or gamma irradiation, which was further confirmed by scanning electron microscopy analysis.

Human embryonic stem cell derived hepatocytes replated on gamma radiated PU 1 34 coated glass cover slips displayed a threefold increase in CYP three a activity over cells replated on UV radiated PU 1 34 coated glass cover slips. These observations demonstrate the gamma radiation is the optimal sterilization technique. While attempting this procedure, it’s important to remember to check the homogeneity of the coating between different spin coating CLOs, And don’t forget that working with chemical solvents instrumentation can be extremely dangerous.

Applications such as wearing a safety goggles should always be taken while performing this procedure.