Tag Archives: cell culture

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.

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…

Laboratory, Project

Building a Stock of PHGL Tumour Baby Cells ….slowly

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My Tumour Baby cells still remain very sluggish and slow to replicate. I’ve been checking in regularly to chart their growth.

Tumour Baby Cells 17/09/21

Tumour Baby Cells 17/09/21

Brightfield microscope images of PHGL TB Cell Growth 17/09/21

As they have continued to grow, they have started to look less healthy and consistent. They remind me of gamma irradiated 3T3 cells (mouse fibroblasts that have been irradiated to stop replicating).  However, this could also be the result of using media that is not ideal, as we have been using some existing (expired) stock supplies of DMEM while we are waiting for our order to arrive.

Tumour Baby Cells 20/09/21

Tumour Baby Cells 20/09/21

Brightfield microscope images of PHGL TB Cell Growth 20/09/21

By 21/09/21 I decided there were enough cells to split into a second flask and freeze down one vial of cells. This will replenish the vial we used and bring our stock up to three vials.

Tumour Baby Cells 21/09/21

Tumour Baby Cells 21/09/21

Brightfield microscope images of PHGL TB Cell Growth 21/09/21

Since the Stroke team mainly work with HBVPs, I reviewed standard protocols for fibroblasts to determine an optimum freezer mix. The recommendation from a number of sources is to include a higher rate of FBS at 30%, 10% DMSO (anti-free agent) and 70% media with a min. of 1 x 106 cells. I made up a total of 1.5mL freeze mix (including cells).

When I passaged the cells, I added 1mL new media to the cells solution. Since the cells were not 80 – 90% confluent, I decided to split them at a rate of 2/3.  This means that the final freeze mix was: 150μL DMSO, 450μL FBS, 300μL media plus 600μL of cell mix. 1mL of this solution was added to cryovial and placed in a freeze box in the -80 degree freezer to be transferred into liquid nitrogen in the next day or so.

Since the cells were precious, I added the remaining cell freeze mix to a T25 flask with 5mL fresh media. There were also a few stubborn cells in the original T75 flask, (post passage),  so I added 10mL new media to see if any of them might grow.  Finally, the remaining 400μL cell mix (without freeze medium) to a new T75 flask with 10mL media.

At this point I had made up fresh DMEM media with the new batch of media, but decided to ‘wean’ the cells onto the new media at a 50/50 ratio of old to new. I am hoping that the new media will help the growth rate of the cells.

Creative Development, Project

Scaffolds Arrived

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Dietmar’s scaffolds have arrived.

Scaffolds in Express Post BagScaffolds arrived via Express Post on 16/09/21

This means that we can do some tests to see how the HBVPs grow in a matrix.  The scaffolds include some flat and tubular sections for initial experimentation.

MPC ScaffoldsMPCL Scaffolds – tubular structures at varying heights.

Scaffolds in Petri Dish Scaffolds in Petri Dish  – flat square structures. 

I will need to confirm with Dietmar’s group but the notations seem to indicate that the scaffolds are  medical grade polycaprolactone (mPCL) with some tubes including a  calcium phosphate (CaP) coating.

MPCL+CaP CoatingMPCL+CaP coating

MPCL+CaP CoatingMPCL+CaP coating – tube structures.

 

Laboratory, Project

Cell Timing…

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When doing cell culture, one has to remain responsive to the cells themselves. I checked my Tumour Baby cells today, anticipating that they would be ready for passage and freeze down, only to see that they are still a bit sluggish post-thawing.

Tumour Baby Cells

Tumour Baby cells at 16/9/21

They are only about 50% confluent, so I will need to wait until they are 80 – 90% before I split them. I will feed them (i.e. change media) tomorrow, but will likely only be able to passage and freeze them on Monday. I just hope they are at the right stage by Monday morning, as I do not have weekend lab access. This puts pressure on getting the timings somewhat right.  Cells wait for no humans….On a positive note, the new batch of DMEM has arrived so I can make up new optimum media tomorrow.

The HBVPs in contrast are ready for passaging.

HBVP P7

HBVPs P7 at 16/09/21

However, they can hold off for one more day so that they will be passaged, fed and happy over the weekend.

Creative Development, Laboratory, Project

Tumour Baby Cell Growth

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I checked in on the tumour baby cells to see how they are growing. This will also help with an estimation of when they need to passaged and I can make up some more frozen stocks.

Overall, they are looking pretty good 🙂 Yay!

Tumour Baby Cells

Brightfield microscope image of Tumour Baby Cells P1 on 14/9/21

They are still a bit sparse, but will likely be ready for passage/freezing this Thursday. That is perfect timing!  I will hopefully be able to make up 3 – 4 vials plus one T75 flask. I might plate them out sparsely again as they will need to grow Friday/Sat/Sun without passaging.

Laboratory, Project

Fixed Cells

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The HBVP cells in the Petri Dishes were ready for fixing on the 14/09/21. I followed a very basic protocol which involved removing the media and and fixing the cells with 4% Paraformaldehyde (PFA) solution for an hour.

After an hour the PFA was removed (in the fume hood in a special waste container) and 5mL PBS added to keep the cells moist and avoid them drying out. I will  stain them later with H&E stain when Jo-Maree has time to show me through histology.

HBVP Clear Petri Dish Fixed

Fixed HBVP cells in clear Petri dish. Image taken 16/09/21

HBVP Sigil Petri Dish Engraved Fixed

HBVP Sigil Petri Dish Engraved Fixed

Fixed HBVP cells in engraved sigil Petri dish. Image taken 16/09/21

Overall the cells seem quite well preserved. However, the cells in the engraved Petri dish with the sigil for “protection from accidents” had the best outcome….maybe sigils make me more vigilant.