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.
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.