Human retinoblastoma is the most common type of intraocular cancer in childhood. Clinical samples are hard to obtain and mice models cannot form the retinoblastoma. Therefore, in vitro retinoblastoma generation is essential for observing retinoblastoma genesis, proliferation, and growth, and for developing novel therapeutic agents.
The efficiency of this protocol can reach about 100%though the duration for retinoblastoma generation is variable from day 45 to day 120. For maintenance of retinoblastoma one or RB one knockout human embryonic stem cells, culture the gene edited H nine cells in a six well plate coated with growth factor reduced basement membrane matrix and change the medium everyday. To passage the cells, incubate them in EDTA buffer for 3.5 minutes at 37 degree Celsius or five minutes at room temperature.
Culture the cells until they reach 80%confluence. Then, to initiate retinal cell differentiation, remove the medium and rinse the cells with one milliliter of DPBS. Next, elevate the cell colonies by adding one milliliter of Dispase buffer and incubating at 37 degree Celsius for five minutes.
Observe the cell colonies under a microscope. Once the edges of the colonies begin to roll out, aspirate the Dispase buffer and gently rinse the cells once with one milliliter of DPBS. Then add one milliliter of medium one into the well and using a 10 microliter standard pipette tip, cut the colonies into pieces.
Use a cell scraper to scrape the remaining cells in the medium. Harvest the cells by centrifugation in a 15 milliliter tube. After centrifugation, leave around 50 microliters of the supernatant to disperse the cells in the medium.
Then add 250 microliters of growth factor reduced basement membrane matrix and mix by gentle agitation. After mixing, place the 15 milliliter tube with the suspended cells into a 37 degree Celsius incubator. After a 20 minute incubation, the cells and growth factor reduced basement membrane matrix should form a solidified gel.
Next, add one milliliter of medium one to the 15 milliliter tube. Using a one milliliter pipette, pipette the solidified gel two to three times to disperse the clump. Then add another nine milliliters of medium one and transfer the cell suspension into a 10 centimeter cell culture dish.
Incubate the dish at 37 degree Celsius and 5%carbon dioxide. This is considered day zero. On day one, examine the cells using a microscope.
On average, three to four cysts are observed in one piece of gel. On day five, change the medium by collecting the supernatant into a 15 milliliter tube, allowing the cysts to settle at the bottom and replacing the supernatant with fresh medium one. Disperse the cysts in the tube into two 10 centimeter dishes, ensuring at least 300 cysts in each dish.
On day seven, most cysts attached to the dish spread out and form adherent colonies. On day 10, change the medium with fresh medium one, ensuring that all the cysts remain attached to the dish. Observe the cells under a microscope from days 13 to 17 to ensure that the cells are spreading out.
On day 15, after rinsing the cells once with one milliliter of DPBS, add one milliliter of Dispase solution and incubate at 37 degree Celsius. After five minutes, remove the Dispase buffer and gently rinse the cultures with one milliliter of DPBS. Then add 10 milliliters of medium two to each 10 centimeter dish and incubate in a 37 degree Celsius and 5%carbon dioxide incubator.
After 24 hours, adherent cultures spontaneously detach and assemble into retinal organoids. Three days after detachment, collect the cells from the cell culture dish in a 15 milliliter tube. When the organoids settle, remove the supernatant from the tube and transfer the organoids to a new non-adherent Petri dish with 10 milliliters of medium two for the following four days.
A week after the detachment, change the medium to medium three and from this day onward, culture the organoids in medium three. On day 27 of the retinal organoid culture, the optic vesicle architecture is evident and around 90%of organoids display this structure. The first detection of the retinoblastoma occurs on day 45 and it becomes palpable on day 50.
When it grows to day 90, the optic vesicle structures are principally enfolded by the retinoblastoma. The retinoblastoma can now be isolated and cultured further as a cell line. On day 105, more than 80%of the retinal organoids are fully enveloped by the retinoblastoma.
These organoids highly express the proliferation marker Ki67 and the oncogene marker SYK compared to the H nine derived retinal organoids, indicating tumorigenesis. Additionally, high expression of the cone precursor marker ARR3 and photoreceptor precursor marker CRX in the retinoblastoma organoids demonstrates that they originate from cone precursor cells. Inferior and superior results during the three morphological stages of retinoblastoma generation are depicted here.
Differentiated and undifferentiated human embryonic stem cells can be easily distinguished based on their morphology. The undifferentiated cells are chosen for retinoblastoma formation. On day five, the sphere generated should be hollow rather than solid.
The retinoblastoma is derived from the retinal organoids that display optic vesicle architecture. No retinoblastoma is generated from the inferior organoids. While performing the procedure, all tips should be cooled and the basement membrane matrix should be melted at four degrees before adding it into the tube.
This is a key step for this differentiation protocol.
