Tag Archives: tumour baby

Laboratory, Project

Media Change and Cell Maintenance

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While I wait for ethics clearance and team members to return to the lab, I am continuing to culture my cells.

Cell Culture 21/1/22

3 x T75 and 1 x Cut Glass Dish ready for cell maintenance (media change) on 21/1/22.

At this point, I am feeding more confluent cells on a weekly basis (depending on how they look):

T75 Flask #1- 21/1/22T75 originally plated 15/12/21 – Flask #1

T75 Flask #1 - 21/1/22T75 originally plated 15/12/21 – Flask #2

T75 Flask #3- 21/1/22T75 originally plated 7/10/21

Cut Glass Dish - 21/1/22Cut Glass Dish – originally plated 15/12/21

While the cut glass dish is hard to image due to the more uneven surface of the glass, and being contained in a larger Petri Dish, there seems to be a good level of cell growth. Perhaps this will result in better staining in the next round.

I still have four other T75s. However, since these are less dense in cell numbers, I am limiting media change to reduce ongoing maintenance costs and media usage:

T75 P4T75 originally plated 7/10/21 and passaged 16/11/21 – Flask #1

T75 P4 - Flask #2T75 originally plated 7/10/21 and passaged 16/11/21 – Flask #2

T75 P5T75 originally plated 15/12/21 – Flask #3

I must admit that I am continually amazed that the original flask of cells plated by Jo-Maree in August last year (and passaged a month later) still has viable cells in it. While much of the T75 flask area is pretty sparse with cells, there are some larger clusters including a growing bunch of elongated fibroid-like cells:

Org plated cells 1T75 originally plated 10/09/21 showing sparse number of cells with evidence of previous cell movement and presence in ‘ghost trails’.

Org plated cells 2T75 originally plated 10/09/21 showing small cluster of cells with evidence of previous cell movement and presence in ‘ghost trails’.

Org plated cells 3T75 originally plated 10/09/21 showing larger cluster of fibroid-like cells.

They are easy to miss, but I’ve made a note to monitor their progress and see how they proliferate. Again…it just takes one mutant to get a cell line going!

 

Laboratory, Project

Review of Cell Flasks

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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 15/12/21Light 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

Tumour Baby P3 - 13/01/22Light 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 15/12/21Photograph 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 16/12/21Light 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 13/1/22Light 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 15/12/21Photograph of PHGL Tumour Baby P5 in T75 flask #2 plated on 15/12/21.

Images taken 16/12/21:

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

Tumour Baby Flask 2 P5 16/12/21Light 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 13/1/22Light 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

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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 - 9/10/21Light 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 FlaskPhotograph of P1 PHGL TB flask originally plated out by Jo-Maree on 10/0921. 

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

Tumour Baby P2 23/09/21Light 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 13/1/22Light 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 MovementLight 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

Extent of death post-holiday – Cut Glass and Petri Dishes

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Following more detailed review of cell flasks, there are some hardy ‘survivors’ of my lab-free holiday.

Cut Glass CellLight microscope image of cut glass dish with media containing dead cell debris and evidence of a small number of surviving cells. 

After removing the old media, it was easier to see the remaining cells:

Cell Survivors - Cut Glass

Light microscope image of cut glass dish in PBS showing evidence of a small number of surviving cells. 

This image shows even more evidence of cell survival:

Cell Survivors - Cut GlassLight microscope image of cut glass dish in PBS showing further evidence of a small number of surviving cells. 

 Cell Survivors - Cut GlassLight microscope image of cut glass dish in PBS with likely cells circled. There are a few additional potential cells visible, but I have only circled the most obvious. 

To get an even better sense of survivors, I will fix (in 4% PFA) and H&E stain 2/3 of the cut glass dishes. The flatter cut glass dish and Petri dish (with more potential for cell survival will be maintained in the incubator to see how they fare over the next week).

Petri Dish Light microscope image of Petri dish with fresh complete media with live cells. 

I will also fix and stain the glass vessels. It is less likely that these will yield anything interesting, but it will help me troubleshoot how to do the protocol with the tiny openings – it is very difficult to effectively remove the media – even with a 20ul pipette and tip 🙁

 

 

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, 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

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.