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.
Microscope image of cells after transduction on 13/4/22. Larger cells indicating viral uptake are outlined in orange.
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.
Microscope image of a large and speckled cell indicating that it has likely taken up the virus. Over the next few days, this cell may start to change and attach to the base of the culture vessel.
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.
Virus vials on ice and in the biosafety hood ready for transduction of PBMCs
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.
Light microscope image showing evidence of dead cells in the previous well. This illustrates that I was very successful at transferring all cells during the media change.
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)!
Light microscope image of PBMC Cells taken on 10/4/22
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.
PBMC Cells on 8/4/22 prior to media changePBMCs 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!
At the UTAS Medical Science Precinct, we are fortunate to have experienced phlebotomists on site to undertake the blood collection process.
Blood Collection
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.
Image documenting first attempt to find a viable vein for blood collection.Image documenting successful collection of blood from right arm.
PBMC isolation
Blood sample in lab.
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.
Blood added to gradient medium ready for centrifugation.
The tubes were then spun in the centrifuge for around 40 minutes which separates cell types and plasma in the sample.
Image of tube post centrifugation showing layers of separated blood. The PBMC layer is highlighted as a thin section between yellow plasma and clear separation medium.
Following centrifugation, the PBMC layer was removed and washed resulting in a small pellet of PBMC cells.
PBMC collection and resulting cell pellet.
The pellet was resuspended in media and a cell count performed to determine the number of viable cells.
Image of densely packed PBMCs viewed under the light microscope.
Following a cell count (using the automatic cell counter), there were around 5 million cells per mL in suspension.
Cell count showing cell count including live/dead cell percentage
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.
Transferring PBMCs to 48 well 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.
After a month of observation and some hands-on iPSC maintenance my training in reprogramming and stem cell culture is largely complete. I am still no iPSC ninja like Ash, but I have a good grasp on the basic processes involved. This means that we can now move forward with reprogramming my own blood cells.
The blood collection is set for the 7th April 2022. This will enable Ash to oversee the PBMC culture and transduction before he goes on holiday over the Easter Break. I will need to stick around, of course to maintain the cultures and wait for attachment (with help from the wonderful Dr Ariane Gelinas-Marion).
The basic schedule is outlined below:
Wednesday 6th April – Prep PBMC media
Thursday 7th April – Blood Collection and PBMC Isolation
Friday 8th April – PBMC media change
Sun 9th April – PBMC media change and cell count
Mon 10th April – PBMC media change
Tuesday 12th April – Add virus – PBMC media
Wednesday 13th April – Transfer to Matrigel Plate with PBMC media
Thursday 14th April – PBMC Media top-up
Saturday 16th April – PBMC Media top-up
Monday 18th April – PBMC Media top-up
Wednesday 20th April – Check for attachment – if attached top-up with Reprogramming Media.
Friday 22nd April – Media Change
Sunday 24th April – Media Change
According to the CytoTune 2.0 Sendai Reprogramming Kit the following basic timeline applies:
Image of basic timeline for PBMC reprogramming using the Cytotune Sendai Reprogramming kit – via ThermoFisher manual.
However, Ash indicated that the full process is likely to take 3 months with a 7 day a week maintenance requirement. While this is pretty heaving going, the actual work should only be 1 – 2 hours per day which makes it manageable. I’m committing – nothing good ever came easy right!
