Notebook

We initiated our first iPSC plate, performing daily media changes while monitoring the cells to ensure proper growth and health. During thawing, we slightly over-pipetted, but the cells successfully clumped by the following day. By the end of the week, we observed strong colony formation.

After passaging our first iPSC plate, we observed fibroblast-like differentiation the following day. ROCK inhibitor (ROCKi) was used during passaging, but we learned that it should only be applied during thawing or when there is a high risk of differentiation. We selected one well of iPSCs that exhibited the least amount of spontaneous differentiation to initiate the organoid differentiation protocol. ReLeSR was used to select undifferentiated cells for organoid formation. We performed aggregate formation (Day 0) and proceeded to Phase I on Day 1, learning the preparation of differentiation media and the collection of organoids. The cell aggregates formed were less than 100 µm in diameter, with uneven edges which improved by the end of phase I.

We received an additional iPSC plate from Dr. Pan’s lab, expanding and passaging it twice successfully, correcting previous issues with ROCKi use. In parallel, we continued the differentiation of our first organoid plate, successfully completing Phase I and proceeding through Phases II and III. Colonies began forming round structures with distinct edges, with Well 6 showing faster growth than the others.

We continued to culture and passage our iPSC plates to build up a stock for future organoid differentiation experiments. In addition, we completed Phase III of our first organoid differentiation and moved to Phase IV. We learned to use a Keyence microscope for imaging, capturing vascularization sprouting in the organoids. However, the organoids were smaller than expected, and we hoped they would grow further in Phase IV.

Phase IV of the organoid differentiation was completed, and we imaged the Day 18 organoids using the Keyence microscope. This confirmed that we successfully generated organoids. We encountered challenges during organoid isolation due to the hydrogel being more watery than anticipated. We identified low pH as the cause and made adjustments for future protocols.

Additionally, we started a new round of aggregate formation but observed that no significant clumps formed upon resuspension in Phase I media on Day 0, prompting us to reconsider our approach.

A second attempt at aggregate formation resulted in the visualization of only a few aggregates (approximately 10 across 4 wells), along with small cell clumps (10-15 cells). Due to the small aggregate size, we determined they would not be viable for further differentiation. A check 12 hours later revealed further dissociation, and no cells gravitated. We continued culturing two iPSC plates, which showed large, healthy colonies with minimal spontaneous differentiation.

We thawed a new iPSC plate to maintain a stock for future differentiation experiments. This plate showed a higher level of spontaneous differentiation, so we changed the media after approximately 20 hours to reduce the concentration of ROCKi, suspecting it may have been the cause.

We initiated a new round of organoid differentiation, this time preparing a much larger batch, filling 54 wells in 24-well ULA plates. Many round aggregates formed, although slightly smaller than expected. The iPSC plate from the previous week showed significant improvement in morphology, with less spontaneous differentiation. We continued culturing and passaged the cells when they reached 80% confluency.

One of our differentiation plates became contaminated with coccus bacteria, with wells turning yellow and cloudy. We bleached the plate and sterilized the incubator. A subsequent iPSC thaw was unsuccessful, resulting in low confluency and no colony formation. Media changes were continued in case of survival.

A new iPSC thaw was attempted, but this plate also became contaminated with coccus bacteria.

We received a healthy iPSC plate from the Center for Cellular Reprogramming (CCR) and began expanding and culturing it. The cells displayed smooth colonies with no spontaneous differentiation or contamination. We initiated aggregate formation using the CCR plate, which resulted in appropriately sized clumps that appeared promising for organoid formation.

We continued expanding and freezing cells from the CCR plate to build up our iPSC stock. We observed a higher yield of aggregate collection via centrifugation and adapted this method for future protocols. However, despite entering Phase I, the aggregates did not increase significantly in size, and many dissociated, preventing transition to Phase II.

We successfully initiated a new round of organoid differentiation using high-confluency iPSCs. Aggregates formed well, with a mix of large and small colonies being maintained. Phase I yielded a high number of promising aggregates through gravitation.

One plate became contaminated, leading us to sterilize our workspace. We continued culturing the remaining plate and froze additional iPSC wells to build up stock. One well was used for aggregate formation, and Phase II of differentiation proceeded successfully, with most aggregates gravitating. Approximately 20-30 organoids were resuspended in Phase II media. However, a significant portion of the cells died before Phase III.

A passage was unsuccessful, resulting in extremely low confluency and signs of iPSC cell death. Media remained pink and largely unused.

We began writing reports and planning contributions to the Wet Lab wiki.

A new iPSC thaw was contaminated. We continued work on lab write-ups.

A new iPSC thaw was initiated, and while the cells formed large colonies, they appeared weak. This plate was also contaminated, likely due to incubator contamination. We initiated a new thaw using a different iPSC line to address this issue.