This methogenetics large quantity of 3D hepatospheres of human pluripotent stem cells using the fine materials and cells of assembly. The main advance of this technique is that it allows controlling the size of the 3D liver spheres, limiting the formation of necrotic centers and loss of phenotype. Demonstrating the procedure with me will be Yu Wang, a post-doctorate in our laboratory.
Prepare to present agarose molds by dissolving two grams of low-melting temperature agarose in 100 milliliters of sterilized distilled water. Carefully heat the mixture in the microwave with interval shaking to dissolve the agarose completely. Add 520 microliters of the melted agarose to a 256 well-format mold and allow it to solidify.
Then, transfer each agarose micro plate into a single well of a 12-well plate. Add 1.5 milliliters of DPBS with calcium and magnesium to each well and pipette up and down to remove bubbles. To prepare the cell suspension, start by aspirating the medium from an undifferentiated culture of human pluripotent stem cells, or HPSCs.
Rinse the cells by adding five milliliters of room temperature DPBS, calcium, or magnesium free and then remove the buffer. Add five milliliters of cell dissociation reagent to the cells and incubate at 37 degrees Celsius for six to eight minutes. Observe cell detachment under a microscope and extend the incubation for an extra one or two minutes if required.
Stop the reaction by removing the cell dissociation reagent and adding five milliliters of fresh MT serve one medium supplemented with 10 micromolar rock inhibitor, Y27632. Pipette up and down several times to dissociate cells. Count the viable cells with the hemocytometer and trypan blue staining in order to calculate the total number of cells needed.
Transfer the desired number of cells to a sterile 15 or 50 milliliter centrifuge tube and centrifuge at 200 times G for five minutes to pellet the cells. Discard the supernatant and re-suspend the cells in MT serve one medium with 10 micromolar rock inhibitor for a final concentration of 2.1 million cells per milliliter. Next, save the cells by adding 190 microliters of prepared cell suspension per agarose micro well and incubating the agarose micro plates at 37 degrees Celsius in five percent carbon dioxide for two hours.
After the incubation, add one milliliter of fresh, warm, T-serve one medium with rock inhibitor to each well. Return the plates to the incubator and leave them there for 24 hours. On the next day, examine the sphere of formation.
Prepare poly-two hydroxyethyl methacrylate or poly-hema coated wells by dissolving two grams of poly-hema in 100 milliliters of 95%ethanol and stirring the solution overnight on a 55 degree Celsius hot plate. On the next day, add 250 microliters of the solution to each well of a 24-well plate and dry the plate overnight in a 60 degrees Celsius oven. Initiate hepatocyte differentiation by carefully removing the MT serve one medium from the previously seeded cells and replacing it with one milliliter of fresh endoderm differentiation medium.
For human embryonic stem cells, refresh medium every 24 hours for three days. It is important to initiate hepatocyte differentiation 24 hours post-seeding to avoid spontaneous cell differentiation. After definitive endoderm induction, switch the medium to hepatoblast differentiation medium for five days.
Replace the medium every two days, performing the last change on the last day of hepatoblast specification. To transfer the hepatospheres into the prepared poly-hema coated wells, wash the cells with hepatocyte maturation medium and then add one milliliter of the medium supplemented with 10 nanograms per milliliter HGF and 20 nanograms per milliliter OSM. Pipette the solution up and down several times to lift the hepatospheres from the agarose micro plate and transfer them to a poly-hema coated well.
Wash the agarose micro plate with one milliliter of the supplemented hepatocyte maturation medium and transfer the medium to the poly-hema coated well. Using a P-100 pipette, carefully aspirate excess medium without removing hepatospheres until there is about one milliliter of medium left in the well. Refresh the medium every 48 hours for 12 days.
If working with embryonic stem cells, switch the medium to hepatocyte maintenance medium at day 20 by removing the maturation medium, washing the cells with maintenance medium, and then adding one milliliter of supplemented maintenance medium. After this, refresh medium every 48 hours. Immediate changes must be performed carefully to avoid serious stress and distortion of this spheric structure.
Staining for hepatocyte in mesenchymal markers revels that the hepatospheres formed using this technique are composed of hepatocyte-like cells surrounding a core of mesenchymal cells. Other proteins typically expressed in hepatocytes are also found on the outer layer of the spheres. Functional analysis of cytochrome P450 enzymes or CYP in 30 day hepatosphere cultures showed that the 3D spheres display respectable levels of CYP activity compared with human primary hepatocytes.
Furthermore, it can be demonstrated that the hepatospheres secrete liver proteins such as Albumin and alfa-fetoprotein. The most important thing to remember for this protocol is to gently pipette up and down the solution containing 3D hepatospheres when transferring them into the poly-hema coated plate. This helps avoid damaging them.
Once mastered, this methodology allows the generation of 3D liver spheres that remain functional over a year in-vitro with multiple applications in decease modeling and drug screening. Looking ahead, this technology could be employed as a platform to develop further entodermal and mesenchymal tissues with complex architectures.
