Tag Archives: lab

Meeting with Ashish Mehta – iPSCs

February 22, 2022 kratz2021 Leave a comment

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

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 Colonies

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.

Gradient Separation

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 10⁶sample. 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.

Laboratory

H & E staining is a bust :(

January 21, 2022 kratz2021 Leave a comment

histology

After spending most of the day in the lab staining up the cut glass dishes and vessels…

Staining set up

… I have emerged victorious-less.

Cut Glass after Staining

Cut Glass after Staining 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.

Cell Vial 1 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.

Cell Vial 2 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.

T25 - Fixed

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.

Laboratory, Project

Review of Cell Flasks

January 14, 2022 kratz2021 Leave a comment

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.

P3 Cell Flask 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:

PHGL TB P3 at 15/12/21 Light microscope image of PHGL Tumour Baby P3 in T75 flask [Org plated 07/10/21] imaged on 15/12/21.

PHGL TB P3 at 15/12/21

Images taken 13/1/22:

Tumour Baby P3 - 13/01/22

Tumour Baby P3 - 13/01/22 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

Tumour Baby P5 Flask 15/12/21 Photograph of PHGL Tumour Baby P5 in T75 flask plated on 15/12/21 prior to holidays.

Images taken 16/12/21:

Tumour Baby P5 16/12/21

Tumour Baby P5 16/12/21 Light microscope image of PHGL Tumour Baby P5 in T75 flask plated on 15/12/21.

Images taken 13/1/22:

Tumour Baby P5 13/1/22

Tumour Baby P5 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

Tumour Baby P5 Flask 2 15/12/21 Photograph of PHGL Tumour Baby P5 in T75 flask #2 plated on 15/12/21.

Images taken 16/12/21:

Tumour Baby Flask 2 P5 16/12/21 Light microscope image of PHGL Tumour Baby P5 in T75 flask 2 plated on 15/12/21.

Images taken 13/1/22:

Tumour Baby Flask 2 P5 13/1/22

Tumour Baby Flask 2 P5 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.

Laboratory, Project

Ghost Movement Trails in Original Tumour Baby Cell Flask

January 13, 2022 kratz2021 Leave a comment

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:

P1 Flask - 9/10/21 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.

P1 TB Flask Photograph of P1 PHGL TB flask originally plated out by Jo-Maree on 10/0921.

Tumour Baby P1 21/09/21 Light microscope image of P1 PHGL TB cells taken on 21/09/21 prior to passage.

Tumour Baby P2 23/09/21 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:

Tumour Baby original flask viewed 13/1/22 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:

Trails of Cell Movement

Trails of Cell 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.

Creative Development, Laboratory, Project

Growing my own cells in Petri Dishes

December 14, 2021 kratz2021 Leave a comment

Following the successful growth of HBVPs in Poly-L-Lysine coated glass Petri dishes, I have enough of my own fibroid cells to repeat the process.

My cells continue to grow so slowly that I should be able to passage them into the Petri dishes and allow them to grow to confluence during the festive season break over 2 weeks . Of course, I need to clear this plan with Jo-Maree. No one else is using the incubator, so it should not be too much of a problem.

As part of this plan, I will be growing my tumour baby cells in 90mm glass Petri dishes and 1 x 90mm crystal dish. As per my previous experiment with HBVP cells, I need to coat the glass surface with Poly-L-Lysine solution to enable cell adherence.

I diluted the Poly-L-lysine solution with sterile MilliQ water (sterilised 14/12/21) to make up 40 mL total (10mL for each 900mm Petri dish x 3, plus 1 x cut glass crystal dish)

6mL PLL + 34mL MilliQ = 40mL PLL Solution

Coating

I added 10mL of the Poly-L-Lysine solution to each dish and then incubated them for an hour. [ The cut glass crystal dish was placed inside a 150mm autoclaved Petri dish to preserve sterility.]

Pll coating glassware Unwrapping Petri dishes and getting ready to coat culture glassware with PLL.

Pll coating glassware PLL coated glassware in Petri dishes ready for incubation.

Following incubation, I removed the Poly-L-Lysine solution and washed the dish with PBS. During cell passage of my confluent flask, I added 1mL of cell solution (from a 10mL suspension) and 5mL media. I placed the cut glass vessels back into a 150 mm Petri dish and into the incubator.

Cut glass dishes with cells ready for incubation Cut glass vessels with cells ready for incubation.

Fingers crossed that they survive the holiday break!

Creative Development, Laboratory, Project

Cut Glass Collection

December 8, 2021 kratz2021 Leave a comment

As part of the residency project, I have started a collection of cut glass items. These were sourced from different second hand shops and build on an existing collection of items used for an exhibition at The Edge at the State Library of Queensland in 2013.

I am particularly attracted to the patterns of the glass. A recurring central motif in many items is a star.

Clear cut glass dish – approx 12cm diameter with central star motif and radiating pattern.

This links to my current interest in deep time including the birth of the universe and emergence of complexity. The glass items also look wonderful when lit from rear. As such, I am considering mounting them over a light source. However, this remains to be seen…

In order to grow cells in the dishes, they need to be sterilised so that they do not carry any bacteria or other organisms that could contaminate my cells.

I am feeling more confident in using the benchtop autoclaves independently so am preparing a batch for sterilisation today. As per previous work, they are placed in autoclave bags and sealed with tape. Once the bags are autoclaved, black lines indicate successful sterilisation.

Cut glass dish and wrapped dish ready for sterilisation.

I have also sources some small glass vials which I am considering integrating into some of the future creative works. There are various shapes that I am planning to test.

Selection of glass vials for cell culture trial including metal closures.

Collection of Glass Items in Autoclave Bags Glass vessels in autoclave bags ready for sterilisation.

Finally, I have also prepared some additional 150mm and 90mm Petri dishes. The large dishes will be used as container vessels for the cut glass dishes to keep them sterile during cell culture.

Petri dishes and other glassware ready for autoclaving Petri dishes and other glass items in autoclave bags ready for sterilisation.

I divided the batch into two runs. As per previous process, I used cycle 6 (134 degrees for 10 min). This enables me to process both glassware and metal. It takes about 10 min for the sterilization process (but extra for cooling to handle materials).

Autoclave instructions with cycle details.

Autoclaved dishes Autoclaved bags containing sterilised Petri dishes.

Autoclaved items stored in lab Autoclaved bags stored in lab area, ready for use.

Laboratory, Project

Lockdown lingers…

October 19, 2021 kratz2021 Leave a comment

Lockdown has lifted, but we have restrictions in place which limits access to lab areas unless absolutely necessary. Jo-Maree has kindly taken over caring duties and will pop in to feed my struggling fibroid cell colonies.

The HBVPs will be put to rest for now with scaffold tests fixed in 4% PFA. We may yet be able to stain them to determine if HBVPs were growing within the structure. Since the scaffolds are optimised for tissue/bone regeneration (and hence bone and tissue cells), they don’t seem to work too well with pericytes – so far anyway.

Since Jo-Maree had a stash of left over vials, we had planned to use Calcein to determine cell viability and visualise the cell growth along the scaffold structure as the scaffolds themselves seem to be non-fluorescent.

Calcein image via APB Biosciences Impressive image of Calcein dye – live cells fluoresce a vibrant green – image via ABP Biosciences.

Since the Calcein dye works on live cells, we will need to reseed the scaffolds when lab-life returns to ‘normal’. This is fine as we will hopefully have enough fibroid cells by then to use for the scaffolds and also undertake fluorescent microscopy – i.e. use antibodies to reveal cell cytoskeleton details (e.g. actin filaments) and DAPI blue-fluorescent dye for nuclei.

Fluorescently labelled cell via Leica Image of fluorescent cells via Leica.

Laboratory

Lockdown…

October 18, 2021 kratz2021 Leave a comment

It has finally happened. We had our first major lockdown in response to the Delta COVID strain. I am hopeful that the estimated closure of non-essential venues (including UTAS) will stand at 3 days. Luckily I was ahead of the game and had already passaged and fed my cells in preparation for Friday teaching. As such, they should be fine until I return on Tuesday.

May the force be with us!

Creative Development, Laboratory, Project

Hematoxylin and Eosin Staining

October 14, 2021 kratz2021 Leave a comment

Jo-Maree finally had some time to go over basic H&E staining procedures. Since my HBVPs are fixed on the base of glass Petri Dishes, the process is much less involved than working with wax embedded specimens.

H&E is a very common stain combination used in histology. Hematoxylin stains nuclei blue-purple
Eosin stains cytoplasm (protein, muscle fibres etc.) pink H & E Stain Protocol Basic H&E staining protocol from Jo-Maree. We only need to follow the staining process.

Stain: washing Petri Dish on bench in Histology Lab at MSP with Erlenmeyer flask containing distilled water for washing.

Prior to adding the Hematoxylin stain, we washed the Petri dishes with distilled water (DW). Usually, we would simply wash the dishes under running water from the tap. However, since rapid water could dislodge the cells from the base of the dish, we have used a beaker to control the water flow. I washed each dish twice to remove PBS and dislodged cells.

Hematoxylin Stain – deep red stain

Contrary to what the name Hematoxylin suggests, the dye is actually naturally derived and comes from the tree Haematoxylum campechianum (Logwood). As such, it is non-toxic and does not need to be added in a fume cabinet. The dye was added to the Petri Dishes for 5 mins, then washed with distilled water.

The next step involved adding ammoniated water (approx 2 – 3 drops ammonia to 400mL distilled water) to the stained cells for 30 secs. This process is referred to as ‘bluing’ and helps change the red – purple hematoxylin to a blue – purple color.

Hematoxylin Stained Dish Cells visible on the base of Petri Dish following Hematoxylin staining.

After washing the Petri Dish thoroughly after ‘bluing’, we added the Eosin stain. Eosin is a xanthene dye and has an intense fluorescent colour.

Eosin stain in Petri Dish.

The Eosin stain only needs 2 mins to stain the cytoplasm and matrix of cells. Following another thorough wash of the dish, we added 95% ethanol and secured the Petri dish lids with parafilm.

For stained sections on glass slides, it is usual to add Xylene (toxic) and a coverslip. In this case, we could either create large scale glass covers (a bit impractical) or clear resin. I think clear resin is the best solution as it would create a barrier and preserve the dyed cells. I am keen to use the fixed cells in dishes as part of sculptural works. However, I will need to check with lab manager David Steele that I am able to remove these fixed cells from the lab.

Creative Development, Laboratory, Project

The struggle is real…

October 12, 2021 kratz2021 Leave a comment

My fibroid cells are still struggling to gain a foothold. I have yet to reach 80 – 90% confluency. We assumed that they are fibroblasts, but the difficulty of growing them in DMEM suggests that they may need different media.

Despite a slow growth rate, on 7/10/21, I passaged my flask of T25 and T75 (approx 70% confluent) at 1:2 to try and increase our stock of cells.

After four days (11/10/21), the cells in the T25 flasks have not grown much and there seemed to be quite a bit of cell debris (i.e. dead cells). I’ve included a few images to provide a better idea of the growth.

T25 Flask 1 - 11/10/21 T25 – Flask 1 P 3, 11/10/21

T25 Flask 2 - 11/10/21 T25 – Flask 2 P 3, 11/10/21

The lag in growth could be a result of these cells growing from the remaining freeze mix. While the DMSO content was very low following plating , exposure to the toxin could have impacted on cell growth and proliferation over time.

In contrast, the T75 flasks seem and doing better. However, growth rate remains slow.

T75 Flask 1 - 11/10/21 T75 – Flask 1 P 3, 11/10/21

T75 Flask 2 - 11/10/21 T75 – Flask 2 P 3, 11/10/21

While we wait for different media to arrive, I added more FBS (20% total) to see if the increase in serum helps stimulate cell growth.

Some common reasons for poor cell growth include:

Starting culture of cells too low in number. This is a possibility, because we thawed and added the fibroid cells directly into a T75. At QUT, we always started primary cells in a T25 to ensure there were enough to stimulate growth.
Incorrect media. This is also a possibility, but it is difficult to determine the best media when we do not know which cell type we are currently working with. We have ordered some DMEM-F12. While this is still optimised for fibroblasts, it may help…plus we need some for the immortalisation and iPSC protocols anyway.
Mycoplasma contamination. The third option is bad. Mycoplasma contamination would require all cells to be destroyed. Regardless, we will need to check if this is an issue.

We could also try bringing up another vial of cells. However, we only have 2 original vials left, so I am a bit cautious using another flask without further trouble shooting.

Plan B

Fortunately, we considered the potential for the fibroid cells to be unviable and have ethical clearance to get new cells via small biopsy. We will continue to try and optimise fibroid cell growth, but it looks like establishing another batch of cells will be more realistic to move the project forwards.

I will follow up with Brad and his colleagues to get the biopsy underway when lockdown (and end of semester marking) is finalised.