Category Archives: Project

Project

Updates in progress…

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Astute readers of my blog may have notices some out of sequence add-ins. This is largely due to ill health last year that limited my capacity to keep up to date with blog entries and direct project progress. ANAT were very understanding and due to health and continued project delays due to COVID (ordering and shipping of reagents and supplies), the project has been extended to May – Phew!

This enabled me to take some dedicated time to get better. It also enables me to move forward with key project milestones in the new year following ethics resubmission.

The result of this is that I am currently updating project documentation. Rather than adding a massive set of posts in one month, I have decided to follow the project timeline with entries posted at the time of action. The aim of this approach is to allow the archived entries to provide a better overview of progress. It will also make it easy for me to refer back to particular moments in terms of cell development. My coding system for flask numbers is pretty bad (e.g. PHGL P3 07/10/21 | 9/11/21 | R2) so having a visual record online will help me keep track of cells and outcomes from trials. This will be particularly important for 2022.

Creative Development, Laboratory, Project

Immunostained Cells

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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

Task Manager

Loading ModeSimple 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 & Phalloidin PHGL Tumour Baby Cells

DAPI & Phalloidin PHGL Tumour Baby Cells

DAPI & Phalloidin PHGL Tumour Baby Cells

DAPI & Phalloidin PHGL Tumour Baby Cells 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 & Phalloidin PHGL Tumour Baby Cells

DAPI & Phalloidin PHGL Tumour Baby Cells

DAPI & Phalloidin PHGL Tumour Baby Cells

DAPI & Phalloidin PHGL Tumour Baby Cells

DAPI & Phalloidin PHGL Tumour Baby Cells

DAPI & Phalloidin PHGL Tumour Baby Cells DAPI and Fluorescein Phalloidin staining of fibroid cells P4  which enables better visualisation of individual cells. 

Creative Development, Laboratory, Project

Immunostaining Protocol

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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 in Fume HoodWorking with 4% PFA in Fume Hood

Prior to imaging, I fixed the cells in 4% PFA:

  1. Remove culture media (discard in waste container with bleach)
  2. Wash cells with PBS (discard in waste container with bleach) x 2
  3. Move cells to fume hood
  4. Add 4% PFA to each well for 15 – 20min at room temp (in fume hood)
  5. Remove 4% PFA solution (discard in PFA waste container in fume hood)
  6. Add PBS (make sure cells are covered or they dry out and produce poor images

IMMUNOSTAINING: At lab bench area

Lab BenchWorking at lab bench in the Stroke Group area

  1. Remove PBS from each well
  2. Add 1mL 0.3% Triton X-100 (a strong detergent) to permeabilize cells (make cells permeable – this allows the phalloidin stain to enter the cell structure) for 10 min
  3. Make up DAPI (5mL PBS Tween and 1ul DAPI) and protect from light with aluminium cover
  4. Remove Triton X-100 and add 1mL DAPI solution to each well and incubate at room temp (with aluminum cover to protect from light) for 5 minutes
    Aluminium Cover
  5. Make up Flouroscein Phalloidin (1mL PBS sand 2μL Flouroscein) and protect from light
  6. Remove and discard DAPI solution
  7. Add 1mL PBS 0.1% Tween to each well for 5 min then discard x 3 (i.e. wash with PBS Tween x 3)
    PBS Tween
  8. Add Phalloidin stain to each well.
  9. Incubate at room temp for 1 – 2 hours
  10. Remove Phalloidin solution
  11. Wash with PBS Tween x 2
  12. Add 1mL PBS in each well
  13. Cover with aluminum foil to protect from light.
  14. Cells are ready for imaging. They can be stored in the fridge (with aluminium foil cover) until ready.

 

Creative Development, Laboratory, Project

Growing my own cells in Petri Dishes

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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 glasswareUnwrapping Petri dishes and getting ready to coat culture glassware with PLL. 

Pll coating glasswarePLL 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 incubationCut glass vessels with cells ready for incubation. 

Fingers crossed that they survive the holiday break!

Creative Development, Project

Revised Ethics – Blood cells for iPSCs

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Due to COVID supply issues, we are still having issues sourcing key reagents etc. for the project. As an alternative iPSC protocol, we are now planning on using blood cells. The main reason is that it is a regular and active protocol in the broader lab area with clear in-house expertise. This also works better conceptually for me than harvesting cells from a skin biopsy – after all blood is strongly associated with notions of kinship . It is also nice to move into the footsteps of my dear colleague Dr Trish Adams who used iPSC technology to turn blood cells into heart cells for the project Machina Carnis.

This does entail a further ethics amendment, but since we have prior approval for skin biopsy harvest, I do not foresee any major issues. I hope to submit this before holidays – ready for the new year!

Creative Development, Laboratory, Project

Cut Glass Collection

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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.

Cut Glass DishClear 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 DishCut 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.

Glass VialsSelection 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 autoclavingPetri 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 InstructionsAutoclave instructions with cycle details. 

Autoclaved dishesAutoclaved bags containing sterilised Petri dishes. 

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

Laboratory, Project

Lockdown lingers…

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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 BiosciencesImpressive 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 LeicaImage of fluorescent cells via Leica. 

Creative Development, Laboratory, Project

Hematoxylin and Eosin Staining

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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.) pinkH & 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 StainHematoxylin 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 DishCells 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 StainEosin 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…

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

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

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

T25 Flask 2 - 11/10/21T25 – 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 - 11/10/21T75 – Flask 1 P 3, 11/10/21

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

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

T75 Flask 2 - 11/10/21T75 – 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:

  1. 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. 
  2. 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. 
  3. 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.