Sculpture Planning and Biodegradable Casting Materials

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….

Media Change and Cell Maintenance

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!

 

H & E staining is a bust :(

histology

After spending most of the day in the lab staining up the cut glass dishes and vessels…

Staining set up

… I have emerged victorious-less.

Cut Glass after Staining

Cut Glass after Staining Microscope images of cut glass dishes after H&E staining on 20/01/22 showing scratches on glass surface and no cells. 

There are no cells visible at all – just scratches on the surface of the glass. This is likely due to the very limited number of remaining cells which may have been further dislodged during the washing process.

The glass vials have not fared much better. While there are cells visible, most of them are dead or dried out as it was tricky working with the small opening and 3D surface area.

Cell Vial 1Microscope image of glass vial after H&E staining on 20/01/22. The image shows a vast number of dead cells that were not fixed in a live state. 

Cell Vial 2Microscope image of glass vial after H&E staining on 20/01/22. The image shows dried cell remnants. 

The flasks show relatively good fixation of the cells, but the staining is not really visible under the light microscope in the ‘dirty’ lab.

T25 - Fixed

Microscope image of fixed PHGL Tumour Baby Cells after H&E staining on 20/01/22. The image shows intact  fixed cells with very limited evidence of H&E stain.

I think, I will stick to Petri Dishes for the next test as they offer a more consistent surface area to work with.

H & E Staining – Protocol Reminder

Today, I plan to stain the cut glass, glass vessels and T25 Flasks.

Before I head over to the lab, I always review the protocol and make sure I have an easily accessible copy.  While it is simple, I have not done it often enough to remember the process without error.

BASIC H&E STAIN:

  1. Remove PBS
  2. Add Hematoxylin – leave for 5 min
  3. Rinse under running water
  4. Add Ammoniated water for 30sec (2 – 3 drops ammonia  to 400mL distilled water)
  5. Rinse under running water
  6. Add Eosin for 2 min
  7. Rinse under running water
  8. Add 95% Etoh for 30sec with agitation
  9. Add 100% Etoh wash x 3

I still need to check with the lab manager if I they are happy that I preserve the stained cells in resin for removal from the lab.

Reviewing the protocol also ensures that I check materials prior to starting the process – there is nothing worse than starting a protocol only to discover that some of the materials are missing.

Ethics – consent and medical questionairre

The ethics amendment process was not quite as quick as I had hoped with a few requests for revisions:

Revision Requests:

I did not think that I need to include formal participant consent forms since I am both participant and lead researcher. However, the ethics officer indicated that this is now a requirement.

While it has meant more work for me, I can see the value in including a formal consent form and medical questionnaire. It ensures that there is clear consent alongside identification of risks associated with the procedures. It also ensure that I formally declare my fitness (i.e. health) in undertaking the processes listed (skin biopsy and blood collection) and clearly list the ways in which risks could be mitigated (e.g. reduce likelihood of bruising, scarring etc). While I already had a good understanding of these parameters, it was useful to have a formal list and agreement. Completing these forms will also make sure I am prepared for future applications, especially involving external (or internal) participants.

I’ve done my best with the document (with review by Brad) so am hopeful for approval for a Feb start! Luckily, I don’t mind admin/paperwork and find the ethics process interesting 🙂

Review of Cell Flasks

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.

Ghost Movement Trails in Original Tumour Baby Cell Flask

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.

 

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

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 🙁

 

 

Updates in progress…

Astute readers of my blog may have notices some out of sequence add-ins. This is largely due to ill health last year that limited my capacity to keep up to date with blog entries and direct project progress. ANAT were very understanding and due to health and continued project delays due to COVID (ordering and shipping of reagents and supplies), the project has been extended to May – Phew!

This enabled me to take some dedicated time to get better. It also enables me to move forward with key project milestones in the new year following ethics resubmission.

The result of this is that I am currently updating project documentation. Rather than adding a massive set of posts in one month, I have decided to follow the project timeline with entries posted at the time of action. The aim of this approach is to allow the archived entries to provide a better overview of progress. It will also make it easy for me to refer back to particular moments in terms of cell development. My coding system for flask numbers is pretty bad (e.g. PHGL P3 07/10/21 | 9/11/21 | R2) so having a visual record online will help me keep track of cells and outcomes from trials. This will be particularly important for 2022.