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.
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!
B-Day is here! That is, Blood Collection Day.
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.
PBMC isolation
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.
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
Logistics
I had a preliminary meeting with Dr Ash Mehta on Monday (21/2) to discuss the iPSC generation using blood. He uses the ThermoFisher CytoTune-iPS Sendai Reprogramming system and has generously offered to guide me through the cell reprogramming/iPSC generation timeline. The great thing about doing this is that the cells are technical immortal when in a stem cell state. This will enable me to achieve the primary project aims even if immortalisation of primary cells via SV40 does not work.
We followed up again on Tuesday (22/2) to go over the process in more detail and set up basic project requirements including blood collection in collaboration with the clinical research team. As part of this process, Ash introduced me to a lovely phlebotomist who agreed to collect my blood, as well as the Menzies Clinical Research Facility Manager to ensure everyone is informed about the project and correct processes are in place to move forward. After supplying project documentation and confirmation of ethics clearance, I received final sign off from the Chair o CRFMC to proceed with blood collection on 23/2/ – so full steam ahead!
Ash also showed me around his lab and allowed me to view the PMBCs (peripheral blood mononuclear cells) he thawed last week. The cells are cultured in suspension (non-adherent) and are circular in shape.
PBMCs in culture – image courtesy of Ashish Mehta
He also showed me some iPSCs and the difference between stem cell colonies and cells that have started to differentiate.
iPSC colony. Image courtesy of Ashish Mehta
About PMBCs
As part the introduction to cell reprogramming, Ash explained the basics and value of working with blood cells.
Blood is made from a number of different cell types including red blood cells (erythrocytes), white blood cells (macrophages, lymphocytes, monocytes, neutrophils, eosinophils, basophils ) and platelets (thrombocytes). Platelets and red blood cells have no nuclei so they cannot be reprogrammed and only the mononucleated (single nucleus) white blood cells are suitable for the process.
To isolate PMBCs, the blood undergoes gradient centrifugation which separates the blood into layers of cell types via density.
Diagram of peripheral blood separated into different layers including PBMCs ( round cells with a single nucleus: lymphocytes, monocytes, natural killer cells (NK cells), dendritic cells).
The advantage of using PBMCs is that you can tell more readily when the virus has initially successfully reprogrammed cells, as they change from non-adherent to adherent and start forming dense colonies of small cells.
The colonies need to be maintained meticulously as they tend to differentiate in culture (i.e. turn into (uncontrolled) specific cell types).
Ash indicated that when the blood is collected, it should be processed (PBMCs extracted) within a 4-hour window. A vial of blood should yield 4 – 5 million PBMCs, so he suggested that we freeze 4 x vials (1 x 106) as backup and proceed with a single 1 x 106 sample. This will also need careful planning to ensure that I am able to donate and process blood on the same day, plus move forward with the next steps involved.
On Thursday 24/2, Ash has kindly agreed for me to shadow him when he adds the Cytotune 2.0 (Sendai Virus reprogramming system) to the cultured PMBC samples. I’m looking forward to learning more.
While I wait for ethics clearance and team members to return to the lab, I am continuing to culture my cells.
3 x T75 and 1 x Cut Glass Dish ready for cell maintenance (media change) on 21/1/22.
At this point, I am feeding more confluent cells on a weekly basis (depending on how they look):
T75 originally plated 15/12/21 – Flask #1
T75 originally plated 15/12/21 – Flask #2
T75 originally plated 7/10/21
Cut Glass Dish – originally plated 15/12/21
While the cut glass dish is hard to image due to the more uneven surface of the glass, and being contained in a larger Petri Dish, there seems to be a good level of cell growth. Perhaps this will result in better staining in the next round.
I still have four other T75s. However, since these are less dense in cell numbers, I am limiting media change to reduce ongoing maintenance costs and media usage:
T75 originally plated 7/10/21 and passaged 16/11/21 – Flask #1
T75 originally plated 7/10/21 and passaged 16/11/21 – Flask #2
T75 originally plated 15/12/21 – Flask #3
I must admit that I am continually amazed that the original flask of cells plated by Jo-Maree in August last year (and passaged a month later) still has viable cells in it. While much of the T75 flask area is pretty sparse with cells, there are some larger clusters including a growing bunch of elongated fibroid-like cells:
T75 originally plated 10/09/21 showing sparse number of cells with evidence of previous cell movement and presence in ‘ghost trails’.
T75 originally plated 10/09/21 showing small cluster of cells with evidence of previous cell movement and presence in ‘ghost trails’.
T75 originally plated 10/09/21 showing larger cluster of fibroid-like cells.
They are easy to miss, but I’ve made a note to monitor their progress and see how they proliferate. Again…it just takes one mutant to get a cell line going!
After spending most of the day in the lab staining up the cut glass dishes and vessels…
… I have emerged victorious-less.
Microscope images of cut glass dishes after H&E staining on 20/01/22 showing scratches on glass surface and no cells.
There are no cells visible at all – just scratches on the surface of the glass. This is likely due to the very limited number of remaining cells which may have been further dislodged during the washing process.
The glass vials have not fared much better. While there are cells visible, most of them are dead or dried out as it was tricky working with the small opening and 3D surface area.
Microscope image of glass vial after H&E staining on 20/01/22. The image shows a vast number of dead cells that were not fixed in a live state.
Microscope image of glass vial after H&E staining on 20/01/22. The image shows dried cell remnants.
The flasks show relatively good fixation of the cells, but the staining is not really visible under the light microscope in the ‘dirty’ lab.
Microscope image of fixed PHGL Tumour Baby Cells after H&E staining on 20/01/22. The image shows intact fixed cells with very limited evidence of H&E stain.
I think, I will stick to Petri Dishes for the next test as they offer a more consistent surface area to work with.
Despite overall loss of cells, one of my P4 flasks which had a good level of cell growth previously still has a strong number of cells. It also shows ‘ghost trails’ over a prolonged period of culture without passage. The flask was originally plated out at P3 on 7/10/21 passaged on 9/11/21 with remaining cells maintained every week.
Image of PHGL Tumour Baby cells Flask with original plating date (P 3, 07/10/21) and passage date (P4, 09/11/21) recorded.
Images taken 15/12/21:
Light microscope image of PHGL Tumour Baby P3 in T75 flask [Org plated 07/10/21] imaged on 15/12/21.
Images taken 13/1/22:
Light microscope images of PHGL Tumour Baby P4 in T75 flask [Org P3 plated 07/10/21 – passaged P4 on 9/11/21] imaged on 13/1/22 after cell maintenance.
I maintained the cells (media change) on 13/1/22 and will allow them to grow for another week before I passage them and set up new experiments with cut glass and Petri dishes.
Prior to leaving for the festive season, I passaged my more confluent culture plates to establish fresh T75 flasks.
Flask #1
Photograph of PHGL Tumour Baby P5 in T75 flask plated on 15/12/21 prior to holidays.
Images taken 16/12/21:
Light microscope image of PHGL Tumour Baby P5 in T75 flask plated on 15/12/21.
Images taken 13/1/22:
Light microscope images of PHGL Tumour Baby P5 in T75 flask plated on 15/12/21 and imaged on 13/1/22.
Flask #2
Photograph of PHGL Tumour Baby P5 in T75 flask #2 plated on 15/12/21.
Images taken 16/12/21:
Light microscope image of PHGL Tumour Baby P5 in T75 flask 2 plated on 15/12/21.
Light microscope image of PHGL Tumour Baby P5 in T75 flask 2 plated on 15/12/21.
Images taken 13/1/22:
Light microscope image of PHGL Tumour Baby P5 in T75 flask 2 imaged on 13/1/22.
In both flasks here is some cell growth present. However, much less than expected. This may be due to my very low seeding ratio which was deliberate to avoid over-confluence during my absence. I changed the media for both flasks on 13/1/22 and will continue to maintain them to see if there is any further growth.