5 tips and tricks for successful hepatocyte differentiation

The Cellartis iPS Cell to Hepatocyte Differentiation System provides a complete solution for generating functional, hiPS cell-derived hepatocytes within three weeks. We know your time is valuable, so we want to help you maximize the success of your differentiation with this system. Our user manual provides a detailed step-by-step protocol for successful differentiation, but here are some of our tips and tricks for the steps that require the most care.

Cell counting

Using an appropriate seeding density is critical for successful differentiation to hepatocytes. Therefore, please make sure to count the cells correctly. We recommend counting cells using a hemocytometer, such as a Bürker chamber. If using an automated cell counter, you should count the same cell suspension in a hemocytometer chamber to verify the results, and adjust the automated counter settings accordingly.

NOTES:

The Cellartis iPS Cell to Hepatocyte Differentiation System includes the Cellartis DEF-CS Culture System for expansion of undifferentiated hiPS cells, the Cellartis Definitive Endoderm Differentiation Kit for differentiation to DE cells, and the Cellartis Hepatocyte Differentiation Kit for subsequent differentiation to hepatocytes.
When performing differentiation of hiPS cells to definitive endoderm cells (DE), using the Cellartis Definitive Endoderm Differentiation Kit, starting with hiPS cell cultures that are too sparse will result in extensive cell death before Day 4 of the protocol, while cultures that are too dense might lead to reduced homogeneity during the differentiation.
When differentiating the DE cells to hepatocytes, with the Cellartis Hepatocyte Differentiation Kit, starting with DE cell cultures that are too sparse will result in a shorter lifespan and suboptimal performance of the HEP cultures. This is because hepatocytes require cell-cell contacts for survival and functionality. If the seeding density is too high, excess cells will not attach and will be removed in subsequent medium changes.

Regular medium changes

During DE differentiation, we change the medium at about the same time every day (e.g., some time between 10 am and 4 pm) to continuously provide the cells with fresh nutrients and factors. During later stages, delaying the medium change by 10–12 hours is not as critical.

Overlay preparation

When preparing the overlay for Days 14 and 16, make sure the medium is at the correct temperature (16–18°C), and use the medium immediately after adding the 3B overlay component. If the medium is too warm, the overlay will gelatinize/fall out before contacting cells and an intact overlay will not form. If the medium is slightly colder, the cells might contract a bit but should recover.

Overlay formation

Do not touch the cells for 48 hours after adding the overlay. If the cells are moved, there is a high risk that the overlay will form unevenly, which will significantly impact differentiation. Resist the urge to look at them. This means not looking at the cells until the next medium change, so no looking/touching the cells on Day 15 and Day 17 (counting from the start of differentiation from hiPS cells, corresponding to Days 8 and 10 after the start of differentiation from DE cells).

Medium changes after addition of overlay

Be very careful when changing the medium from Day 16 onward (which corresponds to Day 7 after the start of differentiation from DE cells). The overlay should not be disrupted or aspirated at any time. We recommend tilting the plate and gently removing 90% of the old medium (shown below in yellow) using a manual single- or multi-channel pipette, then very carefully adding fresh medium (shown in pink) to the wall of the plate.

If you have any questions before you get started or during your differentiation, don't hesitate to reach out to our technical support team here or by clicking on the live chat button in the bottom right corner of your screen (not available in all regions).

Hepatocyte tech notes

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Directed differentiation to hepatocytes

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