Tag Archives: Cellbabies

It’s passage time again + bonus clones

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:

SK iPSC 19/05/22
Light microscope image of iPSC colony on 19/05/22
SK iPSC 19/05/22
Light microscope image of iPSC colony on 19/05/22
SK iPSC 19/05/22
Microscope image of iPSC colonies ready for passage on 19/05/22

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 🙂

SK iPSC 19/05/22
iPSC colony gridded as part of passage protocol on 19/05/22
SK iPSC 19/05/22
Gridded cell sections lifted for transfer to new Petri dish on 19/05/22

It’s official – they’re iPSCs now!

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

iPSC 06/05/22

iPSC 06/05/22

iPSC 06/05/22

iPSC 06/05/22

iPSC 06/05/22

Svenja’s Plates: iPSC 06/05/22

iPSC 06/05/22

iPSC 06/05/22

iPSC 06/05/22

iPSC 06/05/22

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.

My turn passaging cells

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.

Gridding cell colonies with needle
Slightly ‘scratchy’ gridding of cell colonies with the needle on 05/05/22 viewed at .63 X magnification.


First Passage

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).

Colony with cleaned centre
Colony with cleaned centre ready for selection and passage viewed at 1 X magnification. 
Colony gridded with needle
iPSC-like colony gridded with needle during passage on 04/05/22.
iPSC-like colony gridded with needle during passage on 04/05/22.
Another iPSC-like colony gridded with needle during passage on 04/05/22.

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.

Gently lifting cells with spatula
Gently lifting cell sections with spatula.
Gently lifting cell sections with spatula.
Gently lifting cell sections with spatula.

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.

Lifted cell clusters
Lifted cell clusters ready for transfer to new Petri dish.
Pipette collection of cell clusters
Pipette collection of cell clusters.

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.

Preparing for first passage

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.

Unwanted Cells
Example of undesirable non iPSC-like cell colonies viewed at 4 x magnification.
Bad colony
Bad colony with lots of differentiation and no clear iPSC-like edges viewed at 4 X magnification.

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.

Cell colony with central area removed
Cell colony with central area of overgrown cells removed on 03/04/22 for passage on 04/04/22 viewed at 4 X magnification.


More attachment! Moving towards iPSCs

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.

Good series of R-PBMC (precursor iPSCs) visible in Petri Dish #2 – 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 .

A couple of R-PBMC (precursor iPSCs) colonies visible in Petri Dish #2 on 22/04/22

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.

Attached cells with non-iPSC-like morphology on 24/04/22 in Dish #2
Cells with a more promising iPSC-like morphology in Dish #2 on 24/04/22

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.

Large mostly uniform colony of precursor iPSCs on 28/04/22.
More ‘yucky’ colony of precursor iPSCs with cell differentiation visible along the edges of the colony on 28/04/22.

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).

Range of cell clusters on 29/04/22

First Media Change

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.

PBMC Cells
PBMC Cells on 8/4/22 prior to media change
PBMCs after media change
PBMCs after media change on 8/4/22

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!