Category Archives: Laboratory

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, Laboratory

Immunostaining of fibroid cells

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To gain some more insight into the cellular structure of my fibroid cells, I asked Jo-Maree to help me with immunostaining. Immunostaining refers to a staining method that uses antibodies to stain different proteins and structures within the cell. We are going to start with an easy protocol using two antibodies: DAPI and Phalloidin.

DAPI (4′,6-diamidino-2-phenylindole) is a very common fluorescent blue stain used to reveal the nucleus in cultured cells. It can penetrate the intact membrane of the cell. Thus, it can be used for staining both fixed and live cells.

Microscopic image of stem cells, Hues 9 stained with DAPI (blue)Microscopic image of stem cells, Hues 9 stained with DAPI (blue) by the UC San Diego Stem Cell Program.

BASIC LIVE CELL STAINING PROTOCOL

  1. Add DAPI to the complete culture medium used for cultured cells at a concentration of 10 ug/mL.
  2. Remove culture medium from the cells and replace with the medium containing the DAPI.
  3. Cover cells from light exposure and incubate at room temperature or 37°C for 5-15 minutes, then image.

Direct Addition: According to the biotium protocol, you can also stain cells by adding the dye directly to the cell culture and medium. However, this requires a 10X concentration of dye. 

  1. Add the dye to complete culture medium at 10 times the final recommended staining DAPI concentration – 100 ug/mL..
  2. Without removing the medium from the cells, add 1/10 volume of 10X dye directly to the well.
  3. Immediate mix thoroughly by gently pipetting the medium up and down. For larger well sizes (e.g., 24-well to 6-well plates), the plate can be gently swirled to mix.
  4. Cover cells from light exposure and incubate cells at room temperature or 37°C for 5-15 minutes, then image

STAINING FIXED CELLS/TISSUES:

  1. Add DAPI to PBS at 1 ug/mL.
  2. Add the PBS with dye to cells or tissue sections and incubate at room temperature for at least 5 minutes with covering from light exposure.
  3. Samples can be stored in a lightsafe covering (e.g. aluminium foil) at 4°C after staining and before imaging

From: https://biotium.com/tech-tips/protocol-staining-cells-with-hoechst-or-dapi-nuclear-stains/ with minor edits for simpliticy.

Since we are using phalloidin and fixing the cells prior to imaging, we need to follow some additional step to make the cells permeable.

Phalloidin Jo-Maree did not have any relevant stocks, Natalie kindly sourced a sample from another group. [I am always impressed by the generosity of functioning labs and how groups are happy to share stocks to enable other researchers to move forward.]

We are using Fluorescein Phalloidin as a counterstain to enable the visualisation of actin – a protein found in large quantities in the cytoskeleton and cell muscle fibres. As such, it  plays a vital role in cell muscle contraction and overall cell movement.

Interestingly Phalloidin is a toxin (specifically phallotoxin) derived from Amanita phalloides (death cap mushroom).

Amanita phalloidesImage of Amanita phalloides via Wikimedia Commons. 

Phalloidin binds very well to actin filaments and is therefore very useful in visualising cell structure.

Phalloidin staining of actin filamentsU2OS cells stained with fluorescent phalloidin taken on a confocal microscope by Howard Vindin

STAINING FIXED CELLS

  1. Fix cells in 3–4% formaldehyde in PBS at room temperature for 10–30 minutes.
  2. Remove fixation solution and wash cells 2–3 times in PBS.
  3. Add 0.1% Triton X-100 in PBS into the fixed cells for 3–5 minutes to increase permeability. Then wash cells 2–3 times in PBS.
  4. Add phalloidin-conjugate working solution. Incubate at room temperature for 20–90 minutes.
  5. Add DAPI DNA staining dye at this point.
  6. Rinse cells 2–3 times with PBS, 5 min per wash.
  7. Cover with lightfast material to preserve fluorescence

Adapted from:  https://www.abcam.com/protocols/phalloidin-staining-protocol 

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.

Creative Development, Laboratory

Project Delays

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Unfortunately, we have hit another snag in relation to project progress. While we have ethics clearance to proceed with the cell immortalisation process, the ABM cell immortalisation kits we suggested are currently not available in Australia. Jo-Maree found a supplier in Singapore that has the item and associated reagents in stock but the shipping costs are prohibitively expensive at  $2235. This is because the items must be shipped on dry ice which requires additional processing and handling. There are also likely to be delays due to COVID.

The shipping expense is not the only issue as UTAS would need to organise the paperwork for import. As such Jo-Maree has suggested that we investigate alternative options with Australian suppliers. We are leaving this for the New Year.

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.

Laboratory, Project

Final Sign Off

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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:

  1. Plate the target cells in one well of 6-well plate at density of 1-2 x 105 cells/well.
  2. The next day, take one vial of the concentrated recombinant lentivirus from -80 °C freezer and thaw it on ice.
  3. 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.
  4.  The next day, aspirate medium containing viral supernatant and add the appropriate complete growth medium to the cells and incubate at 37 °C.
  5. 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.
  6. 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…