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
Svenja’s Plates: 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.
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.
Slightly ‘scratchy’ gridding of cell colonies with the needle on 05/05/22 viewed at .63 X magnification.
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 ready for selection and passage viewed at 1 X magnification.
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 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 ready for transfer to new Petri dish.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.
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.
Example of undesirable non iPSC-like cell colonies viewed at 4 x magnification.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 of overgrown cells removed on 03/04/22 for passage on 04/04/22 viewed at 4 X magnification.
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.
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
As part of my reprogramming training, I have now learnt how to maintain iPSC colonies. Maintaining iPSCs is a bit different to standard cell culture, as it involves cleaning the plates (i.e. removing unwanted differentiated cells) or selecting specific colonies or areas as part of the re-plating process.
There are two standard protocols in use. The first method is the simplest and involves using a non-enzymatic dissociation reagent. This operates similarly to Trypsin in standard cell culture and dislodges cells so that they can be transferred to a new culture vessel.
The second (and more time-consuming) method involves using dispase to gently break cell adhesion and then manually selecting the areas for collection. A needle is used to grid the selected colonies into equal(ish) segments. These are then gently dislodged with a spatula and transferred to a new culture vessel.
While the UTAS protocols are not for public dissemination, the Stem Cell Catalogue has a good overview of standard/recommended iPSC maintenance.
Over the last month, I have been actively training in stem cell culture with expert Dr Ash Mehta. The training is important to ensure that I understand key protocols and also am able to demonstrate proficiency in cell culture methods. iPSCs are cultured in antibiotic free media, so contamination is a big risk.
As part of the training, I’ve tagged along to view the key protocols involved in reprogramming and iPSC maintenance. Since we are dealing with donor samples, I am not able to disclose images or details of these cells, but can provide a more general overview of some of the processes undertaken.
We started the training on the 24th February with the aim of allowing me to witness the full reprogramming procedure for current PBMC samples.
Reprogramming timeline screenshot from CytoTune-iPS Kit via ThermoFisher.
PBMC Culture – Step One
As visualised in the basic timeline, the first step involves culturing PBMCs in PBMC media for approx. 3 – 4 days prior to transfection. This ensures that they are healthy and growing well prior to reprogramming i.e. dividing, standard in appearance and free from any contamination. PBMCs have a small circular appearance. When they are ‘happy’ they also tend to group together in small clumps.
As outlined in my previous post, PBMCs refer to Peripheral Blood Mononuclear Cells. They are good to use for iPSC generation as it is easy to determine success as reprogrammed cells shift from circular non-adherent cells (free floating in media) to attached cells (growing on the base of the culture vessel).
Transduction [Adding Virus to Reprogram the Cells] – Step Two
After 3 – 4 days of PBMC culture, the cells are ready for transduction. This involves adding a set volume of three engineered Sendai viral vectors to the PBMC cell cultures. This is calculated via the following equation outlined in the CytoTune-iPS Manual:
The viral vectors are used to deliver and express specific genetic segments that effectively reprogram the somatic (differentiated non-germ cells) into iPSCs. The viral vectors are stored in a -70 freezer and must be kept on ice during the addition procedure.
Following the addition of the virus, the cells are incubated at 37°, 5% CO2 for 24 hours. It is important not to disturb them during this crucial stage.
24 hours following transduction, the media containing the virus is removed and the Reprogrammed Cells (RPs) are maintained in fresh PBMC media. Transfected cells often appear larger with dark spots in the interior of the cell and a ‘wobbly’ outer membrane. Some cells burst due to high viral load.
Side note: The CytoTune system uses a non-transmissible form of the Sendai virus (SeV) as delivery vector. SeV is a murine (mouse/rat) parainfluenza respiratory virus from the Paramyxoviridae family. Even though the virus is regarded as non-transmissible, it is important to work safely and sterilise all pipettes, tubes and culture vessels that come into contact with the virus with bleach. As such, viral work is carried out in a specific lab area with fully trained and authorised personnel.
Transfer to Matrigel Coated Plates and Await Attachment – Step Three
On Day 3 (post transduction), the cells are transferred to Petri Dishes coated with Matrigel – a matrix used for iPSC adhesion. Once attachment is visible (cells adhering to the base of the Petri Dish), the media can be shifted from PBMC media to a Stem Cell Reprogramming media such as StemPro or ReproTeSR Feeder-Free Reprogramming Medium.
By day 7 – 9, there should be stem cell like colonies visible. These colonies are circular with small and uniform cells. At this stage, the media is gradually changed to iPSC media, such as mTeSR plus or Essential 8 Media.
Side note: The media is expensive compared to standard cell media such as DMEM, so it is important to plan out culture protocols carefully to avoid waste.
Colonies Emerge – Step Four
By around day 12, iPSC colonies are ready for selection and expansion. They will need to be maintained meticulously to avoid differentiation. This involves regular passaging (splitting) coupled with the removal of differentiated cells to ensure iPSC colonies are preserved.
