All posts by kratz2021

Nothing is ever easy…

After shadowing Jo-Maree and the students in the lab, I feel pretty confident in moving forward with some actual cell work. We are just waiting on the delivery of new cell culture media. There is a bit of a delay with some orders due to the COVID lockdowns in NSW and VIC.

I did receive my glass Petri dishes (90mm and 1500mm) to do some initial tests and had a go at laser engraving them. I usually work in Perspex, so getting the settings just right is unfortunately always a bit of trial and error.

We decided to start with the simple ‘target’ engraving which would enable us to test different engraving depths and see if the graphic should use raster for hatched areas (circles) and vector for lines.

Graphic for Laser Engraving

Graphic for laser engraving shown in Rhino 3D.

We need to leave room for the laser head to engrave so the graphic will be centred in the dish with a 25mm boundary. We also had to ensure that each element was on a separate layer with a colour indicating the particular settings for that graphic element.

We tested some basic settings:

Laser Engraving Settings

Black circle: Raster
Speed: 22
Power: 61
PPI: X
Red circle: Raster
Speed: 42
Power: 61
PP1: X
Green Lines: Vector
Speed: 4
Power: 85
PPI: X

We secured the sides with some metal rods to reduce the likelihood of movement when the air flow is turned on during the laser engraving process.

Laser Set Up
Set up in Laser  with metal bars to reduce movement. 

Laser Engraving ProcessLaser in process.

While the initial engraving seemed promising, the entire graphic was rastered including lines which were supposed to be rendered as vectors. The air flow also moved the dish. This resulted in an initial ‘glitch’ area and, following another shift, an overlaid section.

Engraving Glitch
Laser engraved dish with ‘glitch’ pattern. 

It is not too bad, but shows the importance of testing settings and set up never assuming that things will work first time around.

We used the same dish again to test different settings (with four metal bars to hold the dish in place. However,  we found that the  raster setting was the best approach after all  to ensure that the laser creates lines without punching through the glass. We still don’t quite have the settings down, but hopefully the next go will yield more promising results.

Lab meetings including Science in the Pub Preview by Ash Russell

I have been attending weekly Tuesday Lab meetings at the Medical Sciences Precinct (MSP). This is a great way for me to gain insight into the research of group members. It is also wonderful to be immersed in the science lab culture where meetings become a platform to share work in progress and receive critical input from fellow HDR candidates and supervisors. These presentations usually focus on the research projects of particular  members – much of focuses on stroke research and is  is still in development/unpublished and therefore not suitable for sharing beyond the immediate group setting.

Science Meme

Science Meme via ‘Meme Your Science’ Event by Science in the Pub Tasmania Facebook Event

This Tuesday, was a bit of an exception as we got to listen to a preview of a public presentation by Ash  Russell,for the Science in the Pub Series in Tasmania this Thursday [5th August].  The theme is ‘memes’ so speakers have to use memes to convey their ideas. Ash is currently completing their PhD in bioinformatics, so the presentation focused (quite disturbingly) on the issue of scientific validity with reference to factors such as random chance and publication bias. The presentation was really entertaining so I do recommend heading along (noting that the face-to-face event has sold out, but you can stream along via the Facebook page.

Ethics update

We’ve received our first comment from the HREC Chair regarding our ethics application. They raised an important consideration in relation to more clearly outlining some of the ethical implications and questions that the project raises.  Here is my current thinking:

Posthuman Genetic Legacies raises some ethical issues in relation to the ownership and governance of biological materials. The ethical implications of using human biomaterials for scientific and artistic research form part of the investigation and will enable the research team to identify and consider these issues from creative, legal, and scientific perspectives. This initial enquiry will also form the basis for subsequent exploration which will focus more specifically on bioethics with attention to legal and ethical frameworks for the management of use of biological materials.

Some of the key issues and questions include:

  • Ownership and use of biomaterials (cells and tissue) when removed from the body including tensions arising between individual donor, research team and university.
    • Who legally owns biomaterials (cells and tissue) when they are removed from the body?
    • What rights does the original donor (and researcher-participant) legally maintain when working in a team research environment at a university?
    • What current legal and governance frameworks are in place and which aspects may need reconsideration to better accommodate the interests of all stakeholders?
  • IP implications to produce biomaterials in a university environment
    • How is IP negotiated within a university environment when biomaterials are associated directly with an individual researcher as participant?

Once primary cells are successfully immortalised, the research team will  review current policies regarding the use of biomaterials for artistic use in  Australia. To date, most literature on biological art focuses on the conceptual, ethical and theoretical affordances of the practice and basic lab protocols with limited insight into legal and  governance frameworks, especially where commercial and research interests intersect.

While the creation of the Billy Apple® cell line was a success (Hilton 2014), claims regarding the uptake of the cell line as part of the ATCC are difficult to verify as the cell line is currently not listed in the online cell product listing. Creating another artist cell line for uptake into the American Type Culture Collection (ATCC) or alternative distributor will enable researchers to gain insight into the current policies and practices that underpin biomaterials use, storage and distribution. These insights can then be used to compare policy documentation and experiences of research from art and science over the past 20 years. This will occur in the next stage of project development.

During this phase, the research team will also consider how ethical implications shift when moving from a university research setting to a more open/shared or commercial research environment such as the storage and distribution of biomaterials by companies such as the ATCC.  During this stage, questions will address implications for biomaterial distribution and interdisciplinary engagement:

Some of the key issues and questions include:

  • Ownership and use of biomaterials including tensions arising between individual donor, family, research team, universities, and external organisations such as biomaterial distribution companies.
    • Who legally owns biomaterials (cells and tissue) when they become part of a global biomaterials repository?
    • What ethical guidelines currently govern the use of biomaterials in a global setting? Can you and should you control or restrict the use of biomaterials in relation to personal or cultural values?
    • Should extended family members have input into the distribution of biomaterials (and associated personal information)?
  • IP implications to produce biomaterials across disciplinary terrains (who owns IP when research and commercial interests are involved)
    • How is IP negotiated within a university environment and commercial research setting when biomaterials are associated directly with an individual researcher as participant?
  • Privacy issues for disclosure of personal information (personal details and genetic information) in relation to biomaterials use and distribution.
    • What are the potential risks (short term and long term) involved in the disclosure of personal and genetic information in relation to biomaterials in a research setting (used ​and stored within universities) versus commercial research context (stored and distributed by an external company)?
    • What biomaterial information is useful for scientific versus artistic disciplines?
    • What processes should be put in place to protect the privacy of the donor but still provide useful information across disciplinary domains?
  • Communication including public understanding and transparency of regulatory and governance frameworks in biomaterials research across different domains?
    • What is the public perception and research value of establishing formal frameworks for the development of biobanks and biomaterials for use in artistic research?

This stage will involve further ethical clearance and require me to work with the research team to develop a cohesive research plan and identify ways of minimising risk in terms of privacy.

While I am somewhat familiar with the ethics clearance process, I am new to thinking through the implications as participant where personal medical and genetic information and biomaterial may become shared research materials. This is where collaboration with experts from the Centre for Law and Genetics is vitally important. I look forward to hearing from my collaborator Jane (and hope I was not completely off track).

Hilton, C., 2014. The immortalisation of Billy Apple®: an art-science collaboration. Leonardo47(2), pp.109-113.

Ethics in!

Brad has sent through final information for Stage 2 ethics clearance.  We included a skin biopsy protocol in case the fibroid cells are unviable. Unfortunately, when dealing with primary cells, nothing is certain and there is a possibility that the initial cells are contaminated or do not recover from the freezing process. As such, we have included ‘Plan B’ to ensure we can move forward with the project and establish a new batch of primary cells for immortalisation.

The ethics documents were formally submitted through the Ethics Review Manager. Hopefully, I have addressed all project aspects and provided sufficient information for approval.  Fingers crossed!

Lab Training

Today, I undertook my first day of hands-on lab training. To reduce workload for my kind hosts, I tagged along with 3rd year B/Med students. As part of a unit, they are doing a study into the effects of EGCG (Epigallocatechin Gallate) – an active ingredient in Green Tea which may have positive impacts on stroke patients.  For their study, they are testing the EGCG with HBVP pericyte cells.  The protocols covered today include: cell passage, cell counting and preparing 96-well plates with consistent cell numbers.

It was good to get back into the lab and go over the basics again.  Here is a short summary of the basic protocols involved in cell culture (with some additional insights from Jo-Maree in italics).

CELL SUSPENSION

  1. Put on gloves and ensure they cover the sleeve gap of the lab coat. [Gloves are required for working in the lab. Lab glasses are required for general lab areas but are not mandatory when working in a laminar flow cabinet as the glass offers protection].
  2. Turn on UV light in laminar flow hood (10 mins) to sterilise the interior working area. [Some hood have an automatic turn/off function that enables users to easily sterilise after use. Because these hoods are manual, group users tend to sterilise before use]
  3. Turn off UV and turn on light and blower to start the sterile airflow.
  4. Organise equipment, consumables and media (1 x fluid waste vessel, 1 x pipette waste vessel, pipette and pipette gun, pericyte media, PBS– and TrypLE . Spray all items (including gloved hands) with 70% Ethanol before placing in hood. [Do not place cells in front of waste vessels as this increases contamination due to laminar flow.  PBS– refers to phosphate buffered saline. The — is shorthand for PBS without Calcium (Ca2+) and magnesium (Mg2+) . These minerals help cells adhere to the surface of the culture flask. As such PBS — (without Ca2+ and Mg2+) is used when passaging cells (splitting them into additional flasks).  PBS++ (with Ca2+ and Mg2+) is used to promote cell adhesion. TrypLE is a form of trypsin (a digestive enzyme that detaches cells from adhering to the flask surface). TrypLE is milder and works well with HBVP cells.]
  5. Aliquot media from shared stock and place in heating bed. [The lab has a metal heating bed instead of a water bath. This is a new development and was implemented to reduce the likelihood of contamination as water baths (37 degrees, warm and moist) are perfect breeding grounds for bacteria and a known source of potential  contamination in cell culture).
    Metal Lab Beads for media heating‘Water bath’ using metal pellets to warm media to 37°C (body temperature) – very cool
  6. Collect cells from incubator and view under the microscope. Check cells for any contamination or odd changes in morphology.  [The HBVP cells used today were plated on Friday and are at passage 4 (P4) meaning that they have been split 4 times (although full passage numbers are not disclosed by cell suppliers). Today they were approx. 80% confluent (i.e. they have filled approx. 80% of the available space on the surface of the flask). It is important to not let cultures grow to 100%, as this impacts on cell growth and behaviour. Similarly, cells should be maintained at 30 – 40% min as too few cells also impairs growth. The lab also tends to use cells up to P8 or P9 only to ensure reliable results.]

    MicroscopeMicroscope connected to digital capture

    Cells observed under the light microscope
    Microscope view of cells displayed on the computer screen

  7.   Review cells for any contamination or odd changes in morphology.
  8. Spray flask with 70% ethanol and place into laminar flow hood.
  9. Remove nutrient medium [This can be poured off into the liquid waste vessel, if following correct technique.]
  10. Add 5mL PBS– to culture flask using 5mL pipette.
  11. Gently rotate the flask to wash the cells.
  12. Remove (pour off) PBS solution.
  13. Add 5mL TrypLE (trypsin) and place flask in incubator for 5 min.
  14. After 5 min, check that cells have detached.
  15. Add 10mL complete nutrient medium. [The nutrient medium contains fetal bovine serum FBS which neutralises the trypsin and stops cell digestion]. 
  16. Add cell solution to a 15mL tube.
  17. Spin cells in the centrifuge at 200G for 10 min (room temp). [It is important to add a counter balance when using the centrifuge.]
  18. Check that there is a small cell pellet in the base of the tube.
  19. Spray tube with 70% ethanol and return to laminar flow cabinet.
  20. Gently pour off liquid into the liquid waste vessel.
  21. Resuspend cells in 2mL complete nutrient medium.

CELL COUNTING

The cells are now ready for counting using a traditional haemocytometer – a glass slide with two chambers with a grid of 9 x 1mm squares used to count cells.

  1. Set up haemocytometer by adding the coverslip. [It helps to breath on the slip to create a good seal]
  2. Remove 20 μL of cell suspension.  Remove from hood. [You can place this on a piece of parafilm as it does not need to remain sterile.]
  3. Add 20 μL Trypan Blue to the 20 μL cells and pipette gently to mix the two solutions.  [Trypan Blue stains dead cells as the dye can penetrate through the dead cell membrane. This is used as a mechanism to help count viable cells only].  
  4. Add 10 μL to each chamber.
  5. Since HBVP cells are relatively large, count the cells in the large four corner squares. [When counting cells have a system to avoid doubling up on cells i.e. count top and right only of overlapping cells. Also check to make sure cells are evenly dispersed. If they are clumped together, you will need to suspend cells more thoroughly – i.e. mix with pipette]
  6. Add all cell counts from the corner squares together: 127 + 104 + 116 + 133 = 480 ÷ 4 (number of squares to get an average) = 120 x 104 x 2 (due to dilution with Trypan Blue) = 480 x 104. This is 4.8 x 106 (so 4.8 million cells in the 2mL suspension).
    Cell Counting
    Photo of Jo-Maree’s calculations
  7. To make it to 1x 106 add 280μL to the 200μL.

The students set up 96 well plates with 100μL @ 5000 cells per well  as part their protocol.

CELL PASSAGE

  1. Add 1mL of the 1x 106 cell suspension to a new flask.  Add 9mL fresh warm complete medium and gently rotate the flask to disperse cells.
  2. Remove cells from hood and place them in the incubator. [The cells will be confluent within 48 hours]. 
  3. Remove media, used pipettes and waste vessels from the hood.
  4. Place pipettes and pipette tips in biohazard bins. [If bins are full, take to dirty autoclave station and loosely secure bag with autoclave tape.]
  5. Add liquid waste to bleach to kill any remaining cells. [After 24 hours, all organisms are destroyed and the remaining liquid can be safely disposed of.]
  6. Place waste vessels in bleach bucket in sink area. [After 24 hours, these can be washed and left to dry for re-use].

ON A SIDE NOTE:

Poly-L-lysine is the correct coating to promote cell adhesion on glass surfaces. Jo-Maree has some in stock, so I am all set to work with my Petri dishes as part o the initial cell culture training.

Rhino Files

I have started preparing files in Rhino for laser engraving. This is a good Youtube introduction to setting up files with a some pointers for trimming and adding a hatch for engraving surfaces rather than just lines.

This is the start of my file prep in Rhino with dark areas signalling ‘hatched’ sections for surface engraving.

Screenshot of design in Rhino

Once the Petri dishes arrive, I can make time with the CAM technician Murray Antill to organise engraving. Of course, I need to test the settings first to ensure that I use the correct strength for lines vs. engraving. I usually use Perspex, so I will likely need to adapt the settings to suit a different material. The lines may also need further spacing as the laser produces around a .5mm line.

Immortalised cells – another work

As I continue to develop the project, I am looking into other artists working with cell immortalisation protocols.

A recent project involving the immortalisation of primary cells is the work Immortality for Two which forms part of a dual body of work collectively titled I’am by Luís Graça and Marta de Menezes. In contrast to the aims of the Billy Apple® project, this work reflects on the artist de Menezes and scientist Graça’s long-term working and romantic relationship (de Menezes M & Graça 2020).

Immortality for Two

Immortality for Two, Marta de Menezes 2014 – image available via Bioart Society

For more information see: https://martademenezes.com/portfolio/immortality-for-two/ 

de Menezes M & Graça L I’am – Immortality’s Anti-Marta, in Berger, E., Mäki-Reinikka, K., O’Reilly, K. and Sederholm, H., 2020. Art as we don’t know it. Aalto University School of Arts, Design and Architecture, pp. 52 – 53

 

Design prep

I purchased ten borosilicate glass Petri dishes last week in each size [1500mm and 900mm]. This will enable me to do the engraving and materials tests while I wait for ethics approval.

I am going to start with some simple designs that connect to motifs from my previous practice and signal notions of ongoing development and ‘rippling outwards’.  Screenshot of vector ripple design

Screenshot of vector ‘ripple’ design created in Adobe Illustrator.

This design was originally created for The Contamination of Alice #9 as part of the group show Ghost Biologies at Contemporary Art Tasmania in 2016. I feel that a similar pattern could work quite well engraved on the base of the Petri dishes. However, I will need to include some etched ‘shaded’ areas to see if scarring the surface helps with cell adhesion.

To create the new designs, I will try to work directly in Rhino – the software platform used for the laser cutter. Hopefully, this will enable me to create designs will fewer nodes to reduce clean up time and double lines.

Material supplies: Glass Petri Dishes

My primary lab contact is currently on leave so I am using the time to identify materials for experimentation. I am keen to grow and stain my cells on diverse materials (glass, porous and non-porous scaffolds etc). An easy start is to use glass Petri dishes with different coatings to encourage cell adherence. The use of coatings may also enable me to encourage cells to grow in particular patterns.

I’ve done a bit of searching via Researchgate and it seems that common surface coatings to encourage cell attachment to glass include:

  • Hyaluronic acid
  • Poly-D-Lysine – my current choice
  • Fetal Calf Serum
  • Bovine serum albumin
  • Gelatin
  • Fibronectin
  • Laminin
  • Collagen

I will ask for advice at the next lab meeting.

There were also suggestions to etch the glass surface with concentrated nitric acid and then wash and autoclave. At the UTAS School of Creative Arts and Media, we are fortunate to have access to a glass cutter and laser engraver. So instead of using acid, I will use the laser to score a design into the base of a large 150mm Petri dish.

Glass Petri Dish

Glass Petri dish by Lilly M via Wikimedia

Luckily Petri dishes are easy to purchase online. I just need to make sure the dishes are suitable for autoclave sterilisation (e.g.  borosilicate glass rather than soda lime glass).

Chameleon Pigments

While I plan to spend quite a bit of time in the lab, the Synapse residency is also giving me space to experiment with new creative materials that complement the focus of the research. In particular, I plan on moving forward with chameleon pigment experiments – I have been obsessed with them since the start of the year.

Chameleon pigments are essentially powered colours that have an iridescent quality reminiscent of peacock feathers and beetle wings.

Jewel Beetle

Example of iridescent Jewel Beetle, Jarrahdale State Forest, Western Australia, November 2011 by John Tann via Wikimedia Commons. 

What I particularly like about chameleon pigments is that they shift colour depending on the viewing angle. Not only can they shift between two colours (blue-green), some pigments allow for quite a large spectrum shift (blue-violet-red-orange).

My initial interest started with nail polish (even though I never wear it) as there are a fantastic range of chameleon and special effects options available on EBay and other outlets.

Screenshot E-Bay Search

Screenshot of Ebay search for chameleon nail polish July 2021.

While nail polish was a good start, the small volumes are unsuitable for larger projects. Therefore, my search took me to find larger volume options. I was delighted to discover that chameleon pigments are quite common in craft project and there are a number of retailers in Australia and overseas that produce chameleon and metal mica pigments for a wide range of applications.  The price of these products is variable, so I have been testing small quantities of powder from a range of companies including Solar Color Dust and A1 Pigments.

Screenshot of Chrome Dust Pigments

Screenshot of different chrome dust pigments available at SolarColorDust.com

With the Synapse grant, I plan to expand these initial experiments to include automotive products, as chameleon pigments are also available as for custom car painting and detailing in powder, paint and spray form. Perhaps they will even work to highlight fixed cells on glass.

Chameleon Car Paint

A car with chameleon pigment paint finish taken in 2019 by W Fan (cropped image with license plate number removed) via Wikimedia Commons.