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.
We are now into a bit of rhythm with my little iPSC cell babies. We cleaned the cells on Wednesday (11th) and they are ready for the second passage.
Ash’s plate looks great with lots of lovely iPSC colonies:
My plates are less delightful – you can see some ‘schmucky’ areas marked by broken edges and random growth.
Regardless, there are a few really good colonies to select for the next passage.
At this point, we are continuing to use the slightly more time-intensive dispase method as it enables me to choose the best colonies for continued growth.
As you can see my needle technique is improving, but the size is still a little small at times and not quite uniform.
As part of my project documentation, I’ve also fixed the parent plates this passage using 4% PFA. While the fixed cells are not hazardous, we will also coat the cell base with resin so that the vessels can be displayed as part of artwork components if needed.
Since the plates were passaged on 04/05/22 and 05/05/22, the iPSC cell colonies have grown very well.
My cells passaged by Ash plate: 09/05/22 – originally passaged on 04/05/22
Lovely uniform colonies of iPSC cells (viewed at 4 X magnification).
Svenja’s plates: 09/05/22 – passaged on 05/05/22
As you can see the colonies in my plates are smaller as they are a day behind in growth. While there are some good sections of iPSCs, not all of my colonies are as perfect as Ash’s. At least I do know what I am aiming for in the long term.
From now on we will feed (i.e change the media for the cells on a daily basis) and passage the cells once a week. Our basic weekly roster is as follows:
Monday: Check cells and change media
Tuesday: Check cells and change media
Wednesday: Clean plates ready for passage on following day. Change media.
Thursday: Passage cells.
Friday: Wash cells and change media.
I will need to passage the cells at least 10 – 15 times to ensure the that the initial virus used for reprogramming is no longer present.
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.
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).
When we checked our dishes on the 20th April, we noted that one Petri dish (Dish #2) has definite attachment. This means that we can start transitioning to new media. The other dish (Dish #1) is lagging behind. This is not necessarily a bad thing, but it will delay the media transition for a couple of days. Hopefully the extra time will result in beautifully formed colonies 🙂