Laboratory Log

The “reProductive narrative’s” documented research is going to be published on this site as a Laboratory Log document within the UR Institute residency period, 10th January – 12 February 2021.

TUESDAY, 12th January 2021

After collecting menstruation for an on / off period of 24 months, which we stored in a Vitrification Medium at -20 degress Celsius; our first step was to centrifuge the material and to pour Ficoll® Paque Plus in order to be able to separate the Mesenchymal Stem Cells.

Preparation of the sample
Fresh blood should be used to ensure high viability of isolated mononuclear cells. Prepare the sample at 18ºC to 20°C.

  1. To a 10 ml centrifuge tube add 2 ml of defibrinated- or anticoagulant-treated blood and an equal volume of
    balanced salt solution (final volume 4 ml).
  2. Mix the blood and buffer by inverting the tube several times or by drawing the mixture in and out of a pipette.
    Procedure for isolation of mononuclear cells
  3. Invert the Ficoll-Paque media bottle several times to ensure thorough mixing.
    For withdrawal of Ficoll-Paque media by syringe:
    Snap-off the polypropylene cap and insert the syringe needle through the septum (Fig 1).
    For withdrawal of Ficoll-Paque media by pipette:
    Remove the snap-off polypropylene cap. Lift the aluminum ring. Pull off the metal seal. Remove the silver ring.
    Remove the rubber closure. Using aseptic techniques, withdraw the required volume of Ficoll-Paque media.
  4. Add Ficoll-Paque media (3 ml) to the centrifuge tube.
  5. Carefully layer the diluted blood sample (4 ml) onto the Ficoll-Paque media solution (Fig 3).
    Important: When layering the sample do not mix the Ficoll-Paque media solution and the diluted blood sample.
  6. Centrifuge at 400 g for 30 to 40 min at 18ºC to 20°C (brake should be turned off).
  7. Draw off the upper layer containing plasma and platelets using a sterile pipette, leaving the mononuclear
    cell layer undisturbed at the interface (Fig 4 and Fig 5). The upper layer, which contains the plasma, may be
    saved for later use.
  8. Transfer the layer of mononuclear cells to a sterile centrifuge tube using a sterile pipette.
    Washing the cell isolate
  9. Estimate the volume of the transferred mononuclear cells. Add at least 3 volumes (~ 6 ml) of balanced salt
    solution to the mononuclear cells in the centrifuge tube.
  10. Suspend the cells by gently drawing them in and out of a pipette.
  11. Centrifuge at 400 to 500 × g for 10 to 15 min at 18°C to 20°C.
    Note: A centrifugation at high speed increases the mononuclear cell recovery. However, if it is important to also get rid
    of platelets a lower centrifugation speed is recommended (60 to 100 × g).
  12. Remove the supernatant.
  13. Resuspend the mononuclear cells in 6 to 8 ml balanced salt solution.
  14. Centrifuge at 400 to 500 × g (or 60 to 100 × g for removal of platelets) for 10 min at 18°C to 20°C.
  15. Remove the supernatant.
  16. Resuspend the cell pellet in media appropriate for the application.

WEDNESDAY, 13th January 2021

Executing further research on transforming stem cells to EnSc.

FRIDAY, 15th January 2021

Checking the 12 cultures. They are relatively clean and seem to have quite a lot of cell clusters. We haven+t yet established which are alive.

MONDAY, 18th January 2021

Inspecting the cultures and adding growth media.

WEDNESDAY, 20th January 2021

Again, just checking the colour and changing medium … Time for further research

FRIDAY, 22nd January 2021

Setting up bioreactor and splitting the cells into different habitats, further research before Transfection

MONDAY, 25th January 2021

Inspecing the cells, fishing for stem cells, Fluorescence staining – 1st Trial

WEDNESDAY, 27th January 2021

Further Cell Growth inspection, Immunoflourescence Staining Exercise, Preparing Another Culture of Stem Cells for Transfection

Immunofluorescence staining protocol

STEPS:
1.) FIXATION
PFA (For formal dehydration / powder)
1% solution in PBS (at -20 C)
For application, put in hot water until completely transparent
TIP: 10% solution in PBS placed in smaller volumes (Stock)

2.) PERMABILIZATION

methanol at -20 C

Put on the stations (approx. 5 min)

it will evaporate, so you need to put something so that it doesn’t evaporate outside

3.) COLORING

  1. prepare a 1% paraformaldehyde (PFA) solution in PBS
  2. on the cells you put on the slides, drip a few drops of 1% PFA and let it stand at room temperature for 20 minutes
  3. Remove PFA (you can also pipette freely), put clean PBS and leave for a few minutes (and so 3 times)
  4. When you remove the last PBS, put a few drops of pure, ice cold methanol on the cells (be careful not to evaporate, then put a bowl over it) and leave for 5 minutes (no longer) and then wash again 3 times with PBS
  5. You are now ready

4.) MOUNTING THE COVER GLASS ON THE SAMPLE:
Place glasses with a drop of glycerin on the finished colored sample
Glue 4 edges

TUESDAY, 2nd February 2021

Whole day microscpy; EnSc’s from my blood sample culture showing fibroblastoid morphology, Color: Eosin

TRANSFECTION 3rd culture

MEDIA G1 – 199 (MEDIA FOR CELL GROWING):
0.5 L dH2O bottle
Medium 199 (powder): 4.76 g and HEPES 2.98 g put in 0.4L of distilled water, close the bottle and cook for 30 minutes after it starts to steam. Once completely cooled add to the medium: Pen-Strep (100 x stock solution): 500 uL + Amphotericin: 500 uL + FBS:
a) 50 ml if we do 10%; b) 5 ml if we do 1%.

Half of sample 1 was transferred to transformation medium in petri dish 1 + G2 (transformation medium: 2 mL FBS (30%) + 3 mLM 199 (with antibiotics) + 0.1 mL IVM medium) =) placed further in the incubator for a week.

THURSDAY, 4th FEBRUARY 2021

EXTRACTION OF GONADOTROPIN FROM MY URINE (= THAT IS OF A MENOPAUSAL FEMALE) by USING A CHROMATOGRAPHY COLUMN

1st METHOD folowing http://wlu18www30.webland.ch/wiki/Open_Source_Estrogen

2nd METHOD: following a protocol from the 1965 /// the extraction is executed by decantation in methanol. After a urine sample’s volume is determined, the latter is mixed with a 4 x larger volume of methanol and left overnight. The next day it is decanted, the precipitate discarded and the rest of the alcohol solution is evaporated to 80 degrees Celsius with stirring or blowing with air, to accelerate the evaporation of alcohol.

WEDNESDAY, 10th February 2021

Morphological alteration in EnSCs induced by a synthetic follicular-like fluid. Spheroids two weeks after. We’re getting there …

Pic #1: Spheroidal cell No 1. differentiated in the culture medium supplemented with synthetic follicular-like fluid for 2 weeks (Scale bar: 160 μm)

Pic #2: Spheroidal cell No 2. differentiated in the culture medium supplemented with synthetic follicular-like fluid for 2 weeks (Scale bar: 160 μm)

Pic #4: Microscopy

Pic #6: Further labwork regarding extracting gonadotropin from my menopausal urine for further stimulation of differentiated EnSCs

Pic #7: Centrifuging differentiated cell cultures

Pic #8: Differentiated EnSCs Microscopy to locate and catch the spheroid/s

Pic #9: Criovials for storing the cells into liquid nitrogen for future use.

SATURDAY, 13th February 2021

Starting final inspection of transfected cultures to prepare for immunofluorescence staining. Fingers crossed.

MONDAY, 15th February, 2021

My last menstruation collection was executed exactly one year ago, February 15th 2020 and today I discovered that the morphological alteration in my EnSCs induced by the culture medium supplemented with gonadotropin extracted from my menopausal urine was successful! We got the cells!

Bellow: Capturing the cell under microscope

Bellow: Oocyte-like morphology (3 weeks after transfection)

Bellow: Oocyte-like morphology (3 weeks after transfection)

Bellow: Protein surface marker (Anti-DAZL antibody) detecting oocyte proteins on the cell’s zona pelucida

TUESDAY, 16th February 2021

Finalization: putting the cells to cryopreservation 🙂

During the residency we have therefore recreated the protocol published by the International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiaotong University and collaborators in China, optimized and modified it to be conducted using synthetic reagents supplemented with gonadotropin extracted from my menopausal urine for reprogramming my EnSc’s into oocyte-like cells in order to develop and retell the narrative of a less invasive/intrusive alternative to IVF, to bring such technology closer to citizens, to raise their consciousness and to boost scientifc literacy.

Furthermore, the recent events all over the world show us that the female body has been again used as a means of production in these times of populism in which the significance of the nation increases, placing great value on birth rate. Through such social regression the female body has often been again seen as property of the state, law and ideology. We therefore dedicate this project to all the Others out there and through our deeply dedicated hands on biohacking research & practice based process warmly wellcome further formations of strategic alliances to employ our hormones and bodily fluids as agents for utilizing pharmacologycal and technologycal tools as non invasive (bio)technologies.

A further objective of reProductive narratives is to compose and execute science and art DIY workshop format that generates the subject of (post)reproductive body to be more approachable to public knowledge. Through such open-ended format we will engage and interact with citizens through dialogue, exchange of knowledge, as well as through co-development of open questions and their answers.

We are very much looking forward to autumn when the workshop will take place. Stay tuned.