As per usual schedule Wednesday is ‘cleaning day’. This means removing cell build up (in the centre of colonies) and any differentiated cells prior to Thursday passage:
SK PHGL 1.1 P3:
Good colonies:
Before cleaning:
After cleaning:
SK PHGL 1.2 P3:
Good colonies:
Before cleaning:
After cleaning:
SK PHGL P3:
Before cleaning:
After cleaning;
All plates passaged on 19/05/22 have attachment. This means that I now have three plates of my iPSC cells on the go: SK PHGL 1.1 P3, SK PHGL 1.2 P3 [clones] and SK PHGL P3 [mixed plate].
SK PHGL 1.1 P3:
SK PHGL 1.2 P3:
SK PHGL P3:
All look pretty good at this stage – although the mixed colony plate SK PHGL P3 has some mucky bits and differentiation visible.
We’ve continued with our standard schedule which takes us to our regular Thursday passage time. I am happy to report that my plates are looking much better than documented previously:
Since I have nice separated colonies, Ash suggested that I select single colonies from two plates to establish two clones (1.1 and 1.2) in separate dishes along with a third mixed (3 x colony) plate. This feels like another milestone.
Even my gridding technique has improved 🙂
I am proud to report that my passage skills seem to be improving. The next round of colonies from the latest passage (12/05/22) are looking very good (13/05/22).
There is just one outlier that is definitely a NOT an iPSC colony:
We have good attachment from both plates. This is excellent and Ash indicated that we can now officially stop calling them RPs (reprogrammed cells) and refer to them as iPSCs.
My cells passed by Ash: iPSC 06/05/22
Svenja’s Plates: iPSC 06/05/22
As you can see from the images, Ash’s colonies were larger and more uniform. In the future I will need to increase the size of my gridlines and aim for more uniform sizes of cell sections. It’s all part of the learning process. For now, I am just happy that my cells in both plates attached.
It was my turn passaging cells today using the dispase method demonstrated by Ash yesterday. To gain a bit more experience, I passaged both dishes (Dish #1 and parent plate Dish #2). While the process went well overall, I definitely still need a bit of practice with my needle technique.
Today, we need to passage the first, more established plate – Dish #2. The first passage is always a little risky as it takes skill to ensure attachment of the passaged cells will occur. It is also a little more complex, as we have a lower number of colonies due to low number of initial PBMCs (due to my early media wash error).
As such Ash has kindly offered to do the first passage to ensure viable colonies moving forward. I will passage Dish #1 tomorrow as the colonies need a little more time to grow.
We are using the dispase method to passage the cells as this will enable a careful selection of iPSC-like cells only. This involves incubating the cells with the dispase protease to gently dissociate the cells from the coated dish surface. Once the cells are less attached, good iPSC-like cell areas are selected and gridded up into uniformly sized sections using a needle (around 200 – 500 microns in size = 500 – 1000 cells).
Once colonies are gridded into sections, they are gently removed with a spatula. The aim is to maintain the cells in sections so that they will be more likely to attach and grow new colonies.
Once the desired areas have been lifted with the spatula, the cell clusters are swirled together and collected with a pipette for transfer into a new Petri dish.
Once transferred, the cell sections are dispersed throughout the plate to avoid clustering of colonies. At this stage, we will hold on to the parent plate until we are sure that the cell sections have attached.
We’ve been inspecting the cells on a daily basis to determine when to passage (split) the cells into a new culture plate. The first passage is a crucial to establish good colonies and will likely need to happen on Wednesday 04/05/22. Prior to passage, Ash cleaned the plates to remove overgrown central areas of the colony and undesirable differentiated (i.e. non iPSC-like) cells.
The cleaning process involved using a 200μl pipette tip to gently lift/scrape the unwanted cells, followed by a wash and media change to remove the cell debris to ensure that they do not attach to the plate again.
Since the cells showed the first signs of attachment on the 20th of April [Day 8], I monitored the plates on a daily basis to see the emergence of more reprogrammed PBMC (R-PBMC) colonies (precursor iPSCs) forming on the base of the culture dish.
Plate #2 had colonies on the 20th, so there were some great looking cell clusters visible a couple of days later on the 22/04/22.
Petri Dish #1 was slower for colonies to emerge. However, by the 22nd of April, there were a couple of attached cell clumps .
By the 24/04/22, the initial adherent cells were starting to proliferate well. While attachment and cell growth of any kind is always a good sign, we were keenly hoping to see the emerge of iPSC-like cells. These tend to clump together into small circular clusters.
At this point the cells were still maintained in PBMC transition media, but by the 27/04/22, plates were looking good and we started to shift them to iPSC media. By 28/04/22 [Day 16], the cells are almost able to be classified as iPSCs.
A day later on 29/04/22, the colonies were well and truly growing with a mix of large and small colonies (and some undesirable cells types).
As per our scheduled timeline, we performed the first PBMC media change. This involved collecting and spinning the cells, then resuspending them in fresh media. While the process is quite straightforward, it is tricky working in a small well. I have a feeling that I did not mix well enough and may have left too many cells behind in the well.
There is a remarkable difference between the before vs. after photos, even though it is difficult to photograph cells in suspension just after they’ve been passaged. We will see how they are faring tomorrow…
At least, the frozen PBMCs are safely stored away in liquid nitrogen now!