Final Sign Off

September 30, 2021 kratz2021 Leave a comment

We are minor amendments away from final IBC approval to move ahead with iPSC and Cell Immortalisation processes.

I previously compiled a list of possible options based on available kits and associated literature. Brad and Jo-Maree recommended companies with Australian distributors due to delays in International shipping due to COVID. With this in mind, we identified the ThermoFisher
Epi5 Episomal iPSC Reprogramming Kit for reprogramming the fibroid (Tumour Baby) cells into a stem cell like state. The online product listing also has a comprehensive manual which provides clear instruction regarding the required materials and reagents and protocol. Epi5 Protocol

Overview of key steps in the reprogramming process from Epi5 manual.

We can now move forward with ordering the kit and other required/associated elements.

For cell immortalisation Brad also recommended we use a company with an Australian outlet. Fischer Scientific may be the best option as they have a range of Alstem Immortalization Products. We have identified the SV40 T Antigen and hTERT Cell Immortalization Kits as the most applicable for our cells.

Both kits have good product documentation and manuals available via the Alstem Bio website.

My preference is to ue the SV40 T Antigen. The protocol looks deceptively simple:

Plate the target cells in one well of 6-well plate at density of 1-2 x 105 cells/well.
The next day, take one vial of the concentrated recombinant lentivirus from -80 °C freezer and thaw it on ice.
Infect the target cells in a 6-well plate with 4-20 μl/well viral supernatant in the presence of 4 μl TransPlus reagent (ALSTEM, cat#V050). Note: TransPlus reagent is a polycation that neutralizes charge interactions to increase binding between the pseudoviral capsid and the cellular membrane.
The next day, aspirate medium containing viral supernatant and add the appropriate complete growth medium to the cells and incubate at 37 °C.
After 72 hours incubation, subculture the cells into 2 x 100 mm dishes and add the appropriate amount of puromycin for stable cell-line generation.
10-15 days after selection, pick clones for expansion and screen for positive ones. Note: Since the virus-titer will decrease significantly, we recommend that adding 25% v/v virus protection medium (ALSTEM, cat# VF050) into the thawed supernatant before frozen again for future use.

See: https://www.alstembio.com/web/protocol/SV40_T_Antigen_Cell_Immortalization_Kit_Protocol.pdf

I hope it works out as simply as this sounds…

Laboratory, Project

Building a Stock of PHGL Tumour Baby Cells ….slowly

September 22, 2021 kratz2021 Leave a comment

My Tumour Baby cells still remain very sluggish and slow to replicate. I’ve been checking in regularly to chart their growth.

Tumour Baby Cells 17/09/21

Brightfield microscope images of PHGL TB Cell Growth 17/09/21

As they have continued to grow, they have started to look less healthy and consistent. They remind me of gamma irradiated 3T3 cells (mouse fibroblasts that have been irradiated to stop replicating). However, this could also be the result of using media that is not ideal, as we have been using some existing (expired) stock supplies of DMEM while we are waiting for our order to arrive.

Tumour Baby Cells 20/09/21

Brightfield microscope images of PHGL TB Cell Growth 20/09/21

By 21/09/21 I decided there were enough cells to split into a second flask and freeze down one vial of cells. This will replenish the vial we used and bring our stock up to three vials.

Tumour Baby Cells 21/09/21

Brightfield microscope images of PHGL TB Cell Growth 21/09/21

Since the Stroke team mainly work with HBVPs, I reviewed standard protocols for fibroblasts to determine an optimum freezer mix. The recommendation from a number of sources is to include a higher rate of FBS at 30%, 10% DMSO (anti-free agent) and 70% media with a min. of 1 x 10⁶ cells. I made up a total of 1.5mL freeze mix (including cells).

When I passaged the cells, I added 1mL new media to the cells solution. Since the cells were not 80 – 90% confluent, I decided to split them at a rate of 2/3. This means that the final freeze mix was: 150μL DMSO, 450μL FBS, 300μL media plus 600μL of cell mix. 1mL of this solution was added to cryovial and placed in a freeze box in the -80 degree freezer to be transferred into liquid nitrogen in the next day or so.

Since the cells were precious, I added the remaining cell freeze mix to a T25 flask with 5mL fresh media. There were also a few stubborn cells in the original T75 flask, (post passage), so I added 10mL new media to see if any of them might grow. Finally, the remaining 400μL cell mix (without freeze medium) to a new T75 flask with 10mL media.

At this point I had made up fresh DMEM media with the new batch of media, but decided to ‘wean’ the cells onto the new media at a 50/50 ratio of old to new. I am hoping that the new media will help the growth rate of the cells.

Creative Development, Project

Scaffolds Arrived

September 17, 2021 kratz2021 Leave a comment

Dietmar’s scaffolds have arrived.

Scaffolds in Express Post Bag Scaffolds arrived via Express Post on 16/09/21

This means that we can do some tests to see how the HBVPs grow in a matrix. The scaffolds include some flat and tubular sections for initial experimentation.

MPC Scaffolds MPCL Scaffolds – tubular structures at varying heights.

Scaffolds in Petri Dish – flat square structures.

I will need to confirm with Dietmar’s group but the notations seem to indicate that the scaffolds are medical grade polycaprolactone (mPCL) with some tubes including a calcium phosphate (CaP) coating.

MPCL+CaP coating

MPCL+CaP coating – tube structures.

Laboratory, Project

Cell Timing…

September 16, 2021 kratz2021 Leave a comment

When doing cell culture, one has to remain responsive to the cells themselves. I checked my Tumour Baby cells today, anticipating that they would be ready for passage and freeze down, only to see that they are still a bit sluggish post-thawing.

Tumour Baby Cells

Tumour Baby cells at 16/9/21

They are only about 50% confluent, so I will need to wait until they are 80 – 90% before I split them. I will feed them (i.e. change media) tomorrow, but will likely only be able to passage and freeze them on Monday. I just hope they are at the right stage by Monday morning, as I do not have weekend lab access. This puts pressure on getting the timings somewhat right. Cells wait for no humans….On a positive note, the new batch of DMEM has arrived so I can make up new optimum media tomorrow.

The HBVPs in contrast are ready for passaging.

HBVP P7

HBVPs P7 at 16/09/21

However, they can hold off for one more day so that they will be passaged, fed and happy over the weekend.

Creative Development, Laboratory, Project

Tumour Baby Cell Growth

September 15, 2021 kratz2021 Leave a comment

I checked in on the tumour baby cells to see how they are growing. This will also help with an estimation of when they need to passaged and I can make up some more frozen stocks.

Overall, they are looking pretty good 🙂 Yay!

Tumour Baby Cells

Brightfield microscope image of Tumour Baby Cells P1 on 14/9/21

They are still a bit sparse, but will likely be ready for passage/freezing this Thursday. That is perfect timing! I will hopefully be able to make up 3 – 4 vials plus one T75 flask. I might plate them out sparsely again as they will need to grow Friday/Sat/Sun without passaging.

Laboratory, Project

Fixed Cells

September 15, 2021 kratz2021 Leave a comment

The HBVP cells in the Petri Dishes were ready for fixing on the 14/09/21. I followed a very basic protocol which involved removing the media and and fixing the cells with 4% Paraformaldehyde (PFA) solution for an hour.

After an hour the PFA was removed (in the fume hood in a special waste container) and 5mL PBS added to keep the cells moist and avoid them drying out. I will stain them later with H&E stain when Jo-Maree has time to show me through histology.

HBVP Clear Petri Dish Fixed

Fixed HBVP cells in clear Petri dish. Image taken 16/09/21

HBVP Sigil Petri Dish Engraved Fixed

Fixed HBVP cells in engraved sigil Petri dish. Image taken 16/09/21

Overall the cells seem quite well preserved. However, the cells in the engraved Petri dish with the sigil for “protection from accidents” had the best outcome….maybe sigils make me more vigilant.

Creative Development, Laboratory

HBVPs growing well

September 14, 2021 kratz2021 Leave a comment

The Poly-L-Lysine coating has worked well to encourage growth of HBVPs on the base surface of the glass Petri dishes. They look comparable to the T75 Flask cells.

HBVPs growing in Glass Petri Dish
HBVPs growing on base of glass Petri Dish (no engraving) coated with PLL 13/9/21
HBVPs in T75 Flask HBVPs growing in T75 Flask – 13/9/21

Cells are also growing well in the engraved Petri dishes although it is hard to see the cells on the engraved surface.

Engraved Petri Dish
HBVPs growing on base of glass Petri Dish (with engraving) coated with PLL 13/9/21

To better get a sense of where the cells are growing, we will fix them with 4% Paraformaldehyde (PFA) and then stain them with hematoxylin and eosin (H&E). Hematoxylin has blue-purple color and stains cell nuclei. Eosin is pink and stains proteins more generally.

Laboratory, Project

HBVPs ready and on the go!

September 14, 2021 kratz2021 Leave a comment

I am now well into culturing thanks to Jo-Maree’s expert guidance. I am happy to say that it is all coming back to me and I actually feel pretty confident navigating the labyrinthian lab layout and doing routine cell culture.

I also have my own stock of HBVPs in the Stem Cell lab. As you can see they are quite spindly and form little ‘star clusters’ as they grow:

HBVP_P5_090921

Brightfield Microscope image of HBVP cells (P5 – passage no. 5). These cells were passaged on 9/9/21.

These cells grow in Complete Pericyte Medium (CPM) – Pericyte Medium supplemented with FBS, Antibiotics and Growth Supplements. When keeping a lab journal, it is important to record the Lot no. for all items. This allows researchers to track any variations between batches. It is also vital to record the opening date.

Complete Medium

Complete Pericyte Medium – prepared on 9/9/21

The HBVPs grow in standard T75 Tissue Culture flasks in 15mL of CPM (with cell type, passage number, date of passage and researcher initials listed on the vessel).

HBVP cells

HBVP cells (P4 – passage 4) in T75 Flask.

As part of the experimentation process, I prepared three 90mm glass Petri dishes.

I engraved the base of two dishes with a ripple pattern to see if the engraving would impact on the growth/adherence of the cells. Inspired by the awesome work of Whitefeather Hunter (and my previous collaborative work with sigils), one of these dishes also had an engraved symbol to aid protection from accidents.

As you can see below, the engraving looks quite rugged and sharp as it was done by hand. I am curious to see if the cells will grow on this area.

Engraved Glass

Brightfield microscope image of the engraved glass.

To sterilise the dishes for cell culture, they are placed into paper autoclave bags and sealed with autoclave tape. They are then placed into an autoclave that steam sterilises the vessels at high temperature. The black lines on the tape indicate that the process was successful and the correct temperature was reached.

Autoclaved Petri Dish

Single Petri dish in autoclave bag – dated 26/8/21

Most cells do not adhere well to untreated plastic or glass surfaces. As such we added a Poly-L-lysine coating to aid cell adherence.

poly-l-lysine

Poly-L-lysine solution.

The Poly-L-lysine solution was diluted with sterile H2O to make up 30mL total (10mL for each Petri Dish).

Coating

10mL of the Poly-L-lysine solution was added to each dish and then incubated for an hour.

Petri Dishes

After an hour, I removed the Poly-L-lysine solution and washed each dish twice with sterile H2O. I passaged (split) the HBVPs and added approx 1 million cells (in 1mL media) to each Petri Dish and one T75 Flask (plus 14mL media).

Creative Development, Laboratory, Project

Tumour Babies – rise up!

September 13, 2021 kratz2021 Leave a comment

My tumour baby cells are looking good! So far there are no signs of infection which is excellent, excellent news.

Tumour Baby Cells

Tumour Baby cells – thawed 10/9/21 – viewed 13/9/21

They have been growing for a few days and although they are sparse and sluggish, it is common for primary cells to take a while to recover from freezing. The plan is to grow them up and freeze down some more vials. At the moment, we only have 2 x vials in cryostorage, so there is pressure to build up some additional stocks. I anticipate that they may be ready for passaging at the end of the week.

Project

Slight delay due to (non-COVID) cold…

September 13, 2021 kratz2021 Leave a comment

I’ve had a little radio silence of late due to a nasty cold. My COVID test was negative but I still needed to quarantine at home until all symptoms were clear to ensure I didn’t pass it on.

Despite being sick, we have made some good project progress. Jo-Maree and I also presented our work and passions for the Handball ABC Radio Hobart segment with Joel Rheinberger.

HandBall: Jo-Maree