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!
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.
Today, I plan to stain the cut glass, glass vessels and T25 Flasks.
Before I head over to the lab, I always review the protocol and make sure I have an easily accessible copy. While it is simple, I have not done it often enough to remember the process without error.
BASIC H&E STAIN:
I still need to check with the lab manager if I they are happy that I preserve the stained cells in resin for removal from the lab.
Reviewing the protocol also ensures that I check materials prior to starting the process – there is nothing worse than starting a protocol only to discover that some of the materials are missing.
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.
As indicated in my first post-holiday post, my flasks mostly still have live cells – although some of the more confluent flasks also resulted in mass cell death. Interestingly, my first thawed flask of cells – with remaining cells maintained after the first passage – still has living cells after four months of continuous culture:
Light microscope image of PHGL TB cells at 13/1/22 showing a main cluster area of remaining cells.
These cells were originally thawed and plated on 10/09/21. After passage on 21/09/21 the original flask continued to be maintained with a weekly feeding/media change regimen.
Photograph of P1 PHGL TB flask originally plated out by Jo-Maree on 10/0921.
Light microscope image of P1 PHGL TB cells taken on 21/09/21 prior to passage.
Light microscope image of P2 PHGL TB cells (in original flask) taken on 23/09/21 after cell passage on 21/09/21. A few cells remain visible in the flask.
At present, here are only very small clusters of cells remaining:
Light microscope image of P2 PHGL TB cells (in original flask) taken on 13/1/22. The image reveals a ‘sprinkle’ of cells beyond the main cluster.
The rest of the flask is filled with the ghostly trails of cellular movement:
Light microscope images of P2 PHGL TB cells (in original flask) taken on 13/1/22. The image reveals trails of cellular movement and existence.
Since this is pretty consistent with prolonged culture, I am curious to show Jo-Maree to determine what the ‘residue’ is. It is quite poetic to consider the way in which traces remain of different movements and interactions. I have decided to continue to maintain the flask until there are no more viable cells, so it will be interesting to see how these traces evolve.
Following more detailed review of cell flasks, there are some hardy ‘survivors’ of my lab-free holiday.
Light microscope image of cut glass dish with media containing dead cell debris and evidence of a small number of surviving cells.
After removing the old media, it was easier to see the remaining cells:
Light microscope image of cut glass dish in PBS showing evidence of a small number of surviving cells.
This image shows even more evidence of cell survival:
Light microscope image of cut glass dish in PBS showing further evidence of a small number of surviving cells.
Light microscope image of cut glass dish in PBS with likely cells circled. There are a few additional potential cells visible, but I have only circled the most obvious.
To get an even better sense of survivors, I will fix (in 4% PFA) and H&E stain 2/3 of the cut glass dishes. The flatter cut glass dish and Petri dish (with more potential for cell survival will be maintained in the incubator to see how they fare over the next week).
Light microscope image of Petri dish with fresh complete media with live cells.
I will also fix and stain the glass vessels. It is less likely that these will yield anything interesting, but it will help me troubleshoot how to do the protocol with the tiny openings – it is very difficult to effectively remove the media – even with a 20ul pipette and tip 🙁
The lab has a great set-up for fluorescence microscopy which makes imaging quick and easy.
You just need to load the well plate into the machine and set up basic imaging parameters. You do need to image both DAPI and Phalloidin stains, but the software merges the images for you.
Fluorescent Images of Fibroid Cells
Simple graphic interface with presets ready to complete fluorescent microscopy.
As discussed in my previous post on immunostaining, the blue dots indicate nuclei and the green structures reveal the cytoskeleton via binding to actin.
Confluent wells:
DAPI and Fluorescein Phalloidin staining of confluent fibroid cells P4 (although this is potentially misleading as the cells are very slow growing).
Less confluent wells:
DAPI and Fluorescein Phalloidin staining of fibroid cells P4 which enables better visualisation of individual cells.
It is time to complete the immunostaining protocol with guidance from Jo-Maree. I must admit that with holidays looming, my note taking was a bit sketchy. I will need to follow up with Jo-Maree to record the correct details of DAPI and Fluorescein Phalloidin stain. This will ensure that I know how to prepare (and order) stocks in the future.
I already prepared a couple of wells at different cell concentrations ready for staining. We had to delay the protocol, so some of the higher concentration wells are likely a bit over-confluent. It will be interesting to see how they look under the fluorescence microscope.
CELL FIXATION: ‘Dirty’ Biolab
Working with 4% PFA in Fume Hood
Prior to imaging, I fixed the cells in 4% PFA:
IMMUNOSTAINING: At lab bench area
Working at lab bench in the Stroke Group area
