Category Archives: Creative Development

Creative Development, Laboratory, Project

More attachment! Moving towards iPSCs

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Since the cells showed the first signs of attachment on the 20th of April [Day 8], I monitored the plates on a daily basis to see the emergence of more reprogrammed PBMC (R-PBMC) colonies (precursor iPSCs) forming on the base of the culture dish.

Plate #2 had colonies on the 20th, so there were some great looking cell clusters visible a couple of days later on the 22/04/22.

Good series of R-PBMC (precursor iPSCs) visible in Petri Dish #2 – 22/04/22

Petri Dish #1 was slower for colonies to emerge. However, by the  22nd of April, there were a couple of attached cell clumps .

A couple of R-PBMC (precursor iPSCs) colonies visible in Petri Dish #2 on 22/04/22

By the 24/04/22, the initial adherent cells were starting to proliferate well. While attachment and cell growth of any kind is always a good sign, we were  keenly hoping to see the emerge of iPSC-like cells. These tend to clump together into small circular clusters.

Attached cells with non-iPSC-like morphology on 24/04/22 in Dish #2
Cells with a more promising iPSC-like morphology in Dish #2 on 24/04/22

At this point the cells were still maintained in PBMC transition media, but by the 27/04/22, plates were looking good and we started to shift them to iPSC media. By 28/04/22 [Day 16], the cells are almost able to be classified as iPSCs.

Large mostly uniform colony of precursor iPSCs on 28/04/22.
More ‘yucky’ colony of precursor iPSCs with cell differentiation visible along the edges of the colony on 28/04/22.

A day later on 29/04/22, the colonies were well and truly growing  with a mix of large and small colonies (and some undesirable cells types).

Range of cell clusters on 29/04/22
Creative Development

Ethics Clearance Granted

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After a couple of additional amendments including the submission of a medical questionnaire to confirm suitability for giving blood and formal signed consent form, I have formal UTAS ethics clearance to move forward with iPSC generation from blood.

This is a great milestone and means that I can move forward with lab practice. Brad has also kindly put me in touch with Dr Ashish Mehta who is an expert in stem cell culture.

Creative Development, Off Topic, Project

Sculpture Planning and Biodegradable Casting Materials

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I am currently planning out my upcoming show (June 2022) at The Barracks. Michelle from the Derwent Valley Arts Committee indicated that the large courtyard would make a potentially interesting site to include in the show.

This prompted me to ponder how I might create a sculptural work suitable for an outdoor environment – and whether I could re-make a version of ‘Coming to Terms with Being Forgotten’ –  but designed to disintegrate, shed nutrients and enable the growth of other plants and animals. In this way, the work could better reflect the idea of letting go and making way for what comes after.  It would also force me to be more considerate about the materials used in the construction of the work.

Svenja Kratz: Coming to Terms with Being ForgottenSvenja Kratz, Coming to Terms with Being Forgotten, 2020
Fibreglass, polyurethane, plastic, plants, insects, wax, polymer clay, clay, acrylic paint. Original sculpture exhibited at Rosny Barn (2020) and The Barracks (2021). 

This consideration prompted me to revisit the work of Australian artist Jamie North:

Jamie North - SculptureJamie North, Remainder No.4  2016
cement, blast-furnace slag, coal ash, marble waste, living Australian plants
45cm diameter. 

I like the impermanence disintegrating aesthetic and the integration of live plants into the structure.

Some of his larger works also hint at the ephemerality of all things including monuments and other markers of ‘human ingenuity’.

Jamie North SculptureJamie North, Drifting to Void, 2016
Cement, blast-furnace slag, coal ash, marble waste, steel, living Australian plants, 240 x 67 x 67cm

Jamie North’s work also connects to the ‘TerraForm’ sculptures of Robert Cannon – although I must admit that I prefer the more abstracted works.

Robert Cannon - ApolloRobert Cannon, Apollo, concrete, moss and living plants via: Design Swan

Robert Cannon UprisingRobert Cannon, Uprising, concrete, moss and living plants via: Design Swan

The work of Antony Gormley is also always interesting to consider in relation to the human figure.

In the context of this project, I think the work ‘Sense’ offers an interesting option in relation to the idea of an absent body (with the potential of filling of a void with potential growth).

Antony Gormley SenseAntony Gormley, Sense, 1991, concrete 

While concrete is one material option for producing an outdoor sculpture, I really want to find an alternative casting material that would offer better biodegradability alongside nutrient supplements for organism growth and soil improvement.

There seem to be a growing number of more sustainable materials or biodegradable materials available for casting or injection moulding including bioplastics or  Arboform, which manufacturers describe as ‘Liquid Wood’. However, a number of these products seem to be more designed for industrial and product design purposes. As such, I think this project is more suited to raw materials such as compost or a custom mix between a variety of elements (e.g. sand, rocks, compost, concrete, hay) to enable a range of durational unfoldings and both nutrients and potential habitats.

To help with some of the planning, I consulted designer (and UTAS concrete guru) Jouni Jarvela. He suggested that casting would be a good option, but it would be best to test a range of materials on a smaller scale before sizing the design up to a large-scale format. I do love me some design prototyping! 

Following our chat, I will work on a smaller ‘bust’ version which will be easier to manage than a life-size human form. In the interim, he will consider some material options for testing.

I have to say that one of the things I love about making work is seeing where things go after an initial idea is put forward. At this stage, a life-size courtyard sculpture for June seems out of the realm of possibility – however, a series of degrading self-portrait busts may be the alternative outcome. Who knows….

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, 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!