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 🙂
Observing the cells on 13/4/22 (24 hours after the transduction), Ash was confident that the virus had worked. While not scientifically verifiable, one of the potential indicators for success is the difference in size between standard PBMCs and ‘bloated’ PBMCs that suggest the cells have taken up the virus.
In addition to being larger, cells that may have taken on a viral load tend to have a speckled appearance. The bright halo also indicates that the cell is alive.
We transferred the cells to 2 x 60mm Petri Dishes coated with Matrigel. At this point, we just need to top up the cells with fresh media and wait (and hope) for transformation and attachment. We can also refer to the cells as RP (reprogrammed cells) rather than PBMCs.
Despite low PBMC cell numbers, due to my ineffective media change, we were able to proceed with the virus addition as scheduled. Ash is confident that we will still get a few colonies.
First we transferred the PBMCs to a new 24-well plate with fresh media. The reprogramming process, as outlined previously, is deceptively simple and involved adding a specific volume of each viral vector (total of 3 vectors) to the PBMC culture. Thankfully, Ash calculated the miniscule amounts required.
The virus must be kept cold. As such, the 3 vials were collected and kept on ice during the transduction process.
After the virus was added, the PBMC culture was placed back in the incubator for 24 hours.
On Sunday 10th April, I did another media change for the PBMCs. The number of cells is definitely reduced. When I checked the previous well, there were a number of dead cells visible, so I have clearly left a lot of cells behind.
This shows the importance of mixing the cells well and checking that most cells have been transferred during the media change process. I will need to be really careful and follow Ariane’s instructions carefully to avoid further cell losses.
At this stage, we only have around 1.7 x 105 cells (according to the cell count)!
On the positive side, there is no evidence of contamination and apart from low numbers, the cells seem healthy.
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!
At the UTAS Medical Science Precinct, we are fortunate to have experienced phlebotomists on site to undertake the blood collection process.
We started the collection process at around 9:30am. My first vein (left) was difficult to find, even with the use of a fancy vein illumination machine. However, my second arm (right) had a good vein for blood collection resulting in a 5mL sample for processing.
We transferred the blood to the lab and processed the sample straight away to obtain PBMCs.
The first step involved diluting the blood sample with PBS (saline solution) to make it less viscous. This was then gently added to a tube containing Ficoll solution to form a clear layer of blood over the medium.
The tubes were then spun in the centrifuge for around 40 minutes which separates cell types and plasma in the sample.
Following centrifugation, the PBMC layer was removed and washed resulting in a small pellet of PBMC cells.
The pellet was resuspended in media and a cell count performed to determine the number of viable cells.
Following a cell count (using the automatic cell counter), there were around 5 million cells per mL in suspension.
Ash advised that we only need 500, 000 cells for the reprogramming (so around 100μl).
As such, we added100μl cell mix to 0.5mL media a well in a 48 culture plate.
The remaining cells were resuspended in 2mL freeze media, aliquoted into two cryo-vessels (1mL each), and placed in a Mr Frosty Box (freezing container) in the – 70 freezer. They need to be transferred to liquid nitrogen in 24 hours. These frozen cell stocks operate as quality control and as a backup in case there is an issue with the reprogramming protocol (or lab mishap). Fingers crossed for a smooth journey to iPSC babies.