Laboratory, Project

Blood Collection and PBMC Isolation

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B-Day is here! That is, Blood Collection Day.

At the UTAS Medical Science Precinct, we are fortunate to have experienced phlebotomists on site to undertake the blood collection process.

Blood Collection

We started the collection process at around 9:30am. My first vein (left) was difficult to find, even with the use of a fancy vein illumination machine. However, my second arm (right) had a good vein for blood collection resulting in a 5mL sample for processing.

Trying to find a vein for blood collection
Image documenting  first attempt to find a viable vein for blood collection.
Successful Blood Collection
Image documenting successful collection of blood from right arm.

PBMC isolation

Blood Sample
Blood sample in lab.

We transferred the blood to the lab and processed the sample straight away to obtain PBMCs.

The first step involved diluting the blood sample with PBS (saline solution) to make it less viscous. This was then gently added to a tube containing Ficoll solution to form a clear layer of blood over the medium.

Blood sample for PBMC isolation via density gradient centrifugation
Blood added to gradient medium ready for centrifugation.

The tubes were then spun in the centrifuge for around 40 minutes  which separates cell types and plasma in the sample.

PBMC Layer
Image of tube post centrifugation showing layers of separated blood. The PBMC layer is highlighted as a thin section between yellow plasma and clear separation medium.

Following centrifugation, the PBMC layer was removed and washed resulting in a small pellet of PBMC cells.

PBMC Collection and Cell Pellet
PBMC collection and resulting cell pellet.

The pellet was resuspended in media and a cell count performed to determine the number of viable cells.

PBM Cells
Image of densely packed PBMCs viewed under the light microscope.

Following a cell count (using the automatic cell counter), there were around 5 million cells per mL in suspension.

Cell Count
Cell count showing cell count including live/dead cell percentage

Ash advised that we only need 500, 000 cells for the reprogramming (so around 100μl).

As such, we added100μl cell mix to 0.5mL media a well in a 48 culture plate.

PBMC transfer
Transferring PBMCs to 48 well culture plate

The remaining cells were resuspended in 2mL freeze media, aliquoted into two cryo-vessels  (1mL each), and placed in a Mr Frosty Box (freezing container) in the – 70 freezer. They need to be transferred to liquid nitrogen in 24 hours.  These frozen cell stocks operate as quality control and as a backup in case there is an issue with the reprogramming protocol (or lab mishap). Fingers crossed for a smooth journey to iPSC babies.

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