Tag Archives: immortalisation

Project

Immortalisation kit – supplier found

Leave a comment

After a long delay in finding a suitable Australian supplier for the immortalization kit, Jo-Maree identified an Australian supplier for the ABM SV40 Kit. I contacted them on Thurs (17/2) to see if they are able to supply the kit and provide technical support.

The good news is that the kit is available for purchase and shipping. However, I still need to check with the lab manager that lentiviral work is permissible in the lab I’m using at the moment. Once this is confirmed, I can move ahead with a quote.

Laboratory, Project

Final Sign Off

Leave a comment

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…

Creative Development, Laboratory, Project

LAB SCHEDULE

Leave a comment

UPDATED LAB SCHEDULE

Now that the project has the formal go-ahead, I am moving into lab mode and have determined some of the key milestones for the next months.

1: Training & Prep: 1 – 2 weeks

Training with HBVP cells include:

  • Thawing and culturing cells, making media, working in a biosafety cabinet and maintaining sterility, light microscopy
  • Learn to use the autoclave and prepare petri dishes and glass vessels for culture
  • Coat petri dishes and glass vessels with poly-l-lysine for cell adhesion, test with HBVP cells
  • Order media, reagents and kits
  • Submit IBC approval forms

2: Cell culture of fibroid cells – 4 – 8 weeks

  • Thawing and culture – grow up and freeze stocks of cells, light microscopy
  • Ask Dietmar to send 3D scaffolds
  • Grow and fix cells in petri dishes and glass vessels
  • Fluorescent microscopy of cells
  • Scanning Electron Microscopy (SEM) of cultured cells
  • Transmission Electron Microscopy (TEM) of cultured cells
  • Timelapse microscopy
  • 3D cell seeding HBVPs and Fibroid cells – see differences in cell response.
  • Wait for IBC approval

PROJECT: 3 months

3: Cell Immortalisation +

  • Immortalisation of primary fibroid cells via established commercial kit (Applied Biological Materials (ABM) or Alstem cell immortalisation kits)
  • Cell genetic profiling
  • Cell culture of immortalised fibroid cells (optimisation of culture methods for 2D and 3D environments, cellular response and proliferation testing)
  • Grow and fix cells in petri dishes and glass vessels
  • Timelapse microscopy

4: iPSC production

  • Reprogramming of primary cells to generate induced pluripotent stem cells (iPSCs) via established commercial kit (e.g. Epi5™ Episomal iPSC Reprogramming Kit available via Thermo Fisher)
  • Development of Gastruloids, Organoids or Neurospheres (self-organised 3D cell masses)
  • Cell culture of reprogrammed or immortalised fibroid cells (optimisation of culture methods for 2D and 3D environments, cellular response and proliferation testing)
  • Timelapse microscopy
  • If iPSC successful – create neurons and heart cells
Project

Ethics GRANTED! …but one more approval to go…

Leave a comment

We have approval to move forward with the key aims of the project. This is great news as it means I can start working with own cells. I am still a bit precious as there are limited vials, so I will do a couple of weeks of training on HBVPs before I move on to my own cells.

While we can get started on the fibroid cell culture, Brad realised that cell immortalisation will require further Institutional Biosafety Committee (IBC) approval. This is because the process will require the uses of lentiviral vectors. As such, it is considered Notifiable Low Risk Dealing (NLRD) and the committee will need to ensure that we have the appropriate facilities and training in place to move forward.  iPSC cell reprogramming is exempt, but we still need to let the IBC know what we are doing.

The application is due tomorrow, so we had a meeting this morning to go over the protocols and identify the particular kits we are going to use. There are a range of biomedical research supply companies, but the important thing is to make sure that we use an organisation that has an Australian supplier.  hTERT and SV40 T Antigen kits are the best options for our project as they are suitable for a range of  cell types including fibroblasts.  Fischer Scientific have Alstem Immortalisation Kits available, but ABM may also be a good option. They also have a good overview of Cell Immortalisation Protocols for anyone interested in the process.

For iPSC reprogramming, we are going to use the Epi5™ Episomal iPSC Reprogramming Kit by Thermo Fischer. Another group has used this product previously – so we can get tips on how to get the best results.  Lovely Jo-Maree is looking into the best purchasing options. With lead time for purchase and delivery, the products will likely arrive around the same time as final approval.

Project

Cell Immortalisation Products

Leave a comment

In preparation for meeting with Brad and Jo-Maree, I compiled a list of cell immortalisation products. Many of the companies listed are US, so this may impact on availability.

CELL IMMORTALIZATION KITS:

  • ALSTEMBIO: SV40 T Antigen Cell Immortalization Kit 

GENTARGET: SV40 Large T antigen  

GENECOPEIA: Cell Immortalization Reagents 

FISCHER SCIENTIFIC: Alstembio 

CAPITAL BIOSCIENCES: Lentiviruses for Cell immortalisation 

ABM: Cell Immortalisation 

BIOCAT 

CREATIVE BIOARRAY 

Artworks, Creative Development

Immortalised cells – another work

Leave a comment

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