Monthly Archives: October 2021

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. 

Laboratory

Lockdown…

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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

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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.

Creative Development, Off Topic

Sponges as scaffolds?

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We are lucky in Tasmania to be able to travel freely across the state. With winter coming to an end, I saw an opportunity to visit Burnie with some art school colleagues. We witnessed the arrival of some of the first penguins at the Burnie Little Penguin colony for their annual mating and childrearing.

We also glanced some amazing sponges on the beaches in nearby Wynyard.

Wynyard BeachFossil Bluff – Wynyard.

Doctor's RocksDoctor’s Rocks – Wynyard

Marine Sponge texture

Seeing the texture and architecture of different marine sponges on the beach, prompted me to consider whether they have been considered as a scaffold architecture for cell growth.

Sponges from WikimediaDifferent sponges from Wikimedia Commons.

Turns out that yes, there is already a study on whether marine sponges could be used as scaffolds in bone repair.

In vitro Evaluation of Natural Marine Sponge Collagen as a Scaffold for Bone Tissue Engineering

While this has already been done. I think it would still be a nice side experiment to see whether I can grow my cells in a marine sponge scaffold. There are a number of companies that offer cleaned and bleached natural marine sponges for bathing, facial exfoliation and art – although the variety seems usually limited to honeycomb and silk sponges from the Mediterranean.

Sponges

Bag of sponges available from art supply store.

Perhaps the sponges could be used in conjunction with a hydrogel to assist with cell adhesion and proliferation. I think it would be quite lovely to make a self-portrait of ‘me’ as a sponge. Although, I will likely need to use a bioreactor to enable nutrients to reach the interior of the structure.