PBMC Isolation using Lymphoprep

PBMC Isolation using Lymphoprep

Principle of the separation procedure

This is a simple and effective protocol most commonly used to isolate mononuclear cells AKA PBMCs from human blood which was devised back in 1968.

The Lymphoprep Tube  is a sterile tube in that contains the Lymphoprep (separation medium) under a plastic filter disc. This allow the PBS-diluted blood to be poured directly into the tube, while the filter disc prevents any mixing with the separation medium.

The separation principle is largely synonymous to the widely known density gradient centrifugation. On further elaboration, this procedure uses the strong centrifugal force to force components in solution to be "layered" according to their density. According to physics, the higher the density (heavier mass) of a component, the lower portion the component will be found in the tube.

Therefore, the Lymphoprep Tube uses this theory to "purify" and isolate the PBMCs. The Lymphoprep solution is a specially formalized separation medium that has a density of 1.077 g/ml. Since RBCs and polymorphonuclear granulocytes (Neutrophils, Eosinophils) are cells that have a density more than 1.077 g/ml, they will be forced through the filter disc and pellet at the bottom of the tube. As a consequence, the Lymphoprep Solution will be displaced and is "pushed" upwards. The mononuclear cells (PBMCs) - lymphocytes and monocytes are known to have densities lighter than 1.077 g/ml, hence they will be found just above the Lymphoprep Solution and this layer is commonly known as the buffy layer. Lastly, there are other components found in the plasma such as electrolytes, hormones, enzymes and tiny cells known as platelets will be found just above the buffy layer.

Product inset

http://www.axis-shield-density-gradient-media.com/Package%20insert/Lymphoprep.pdf

PBMC Isolation using The Miltenyi Biotec MACS Cell Separation Technology

PBMC Isolation using The Miltenyi Biotec MACS Cell Separation Technology

Background information 

The Miltenyi Biotec MACS Cell Separation Technology is one of the most prominent powerful tool for specific cell separation. This MACS technology is based on MACS Microbeads (Antibodies coupled with Magnectic Beads), MACS Separator and the MACS Columns. MACS MicroBeads are superparamagnetic antibody particles that are approximately 50 nanometers in diameter and are composed of a biodegradable matrix. Furthermore it is manufactured in a manner that does not detrimentally affect nor modify the alter structure, function, or activity status of labeled cells. Hence, it does not interfere with the subsequent downstream experimental processes and is not necessary to remove them from cells after the separation process. 

The MACS separation process occurs within the MACS Columns. The MACS Separator acts as a “magnetic bar” by inducing a strong permanent high-gradient magnetic field into the column matrix. Target cells that are labelled with MACS Microbeads are attracted to the column walls by the magnetic force (Positive Selection). Unlabelled cells (Negative Selection), are allowed to flow and pass through to be collected at the other end via natural gravitational force. Hence, with MACS technology, both Target and non-Target cell fractions can be collected and isolated with high purity to be subjected to downstream experimental procedures immediately. 

* A video link is provided for a more clearer picture of the manual method of this Miltenyi Biotec MACS Cell Separation Technology: http://www.youtube.com/watch?v=6vyj8-OkOKc.
Watch it with your own discretion as research and experimental aims may differ. 

Process of Miltenyi Biotec MACS Cell Separation Technology

Magnetic Labeling 

Highly purified PBMCs can be isolated from whole blood via the procedure in the previous chapter. Subsequently the PBMCs are re-suspended in the appropriate amounts of AutoMACS Buffer depending on individual protocol and incubated with the desired MACS Microbeads that is specific to the antigen on the Target cells. The incubation process is usually atleast 15minutes to a maximum of 30 minutes in the optimal temperature of 4°C usually in a refrigerator.

Next, the sample is then resuspended further with the appropriate amount of AutoMACS Buffer and subjected to washing. This washing step is done by first centrifuging the sample and discard the supernatant to remove any unbound MACS microbeads, leaving a PBMC cell pellet. This PBMC cell pellet is then subjected to the subsequent MACS separation procedure.

Magnetic Separation (Part 1)

After the PBMC cell pellet is resuspended with the appropriate volume of AutoMACS buffer, the cell suspension is then pipetted into the AutoMACS Column to allow it to flow through in a drop-wise manner via natural gravitational force. Target cells that are labelled with the antibody coupled with magnetic beads will get attracted onto the side of the AutoMACS Column via magnetic attraction forces produced by the magnetic separator. On the other hand, non-target cells that are not labelled, will flow through into a collection tube as the negative fraction.

Magnetic Separation (Part 2)

After the first initial cell suspension have fully flowed through, the AutoMACS Column is then subjected to washing to make sure that all non-target cells are washed into the collection tube, leaving a purified positive fraction in the walls of the column. The washing step is a procedure performed simply by adding AutoMACS buffer into the AutoMACS column and leave it to flow through. The number of washing steps and the volume of AutoMACS Buffer varies with different protocol.

Elution of the labelled target cell fraction

After the completion of the washing step, a plunger is then used to “push” all the target cells that are attracted on to the walls of the AutoMACs Column. This is done by first removing the AutoMACS column from the Magnetic Separator, proceeding with pipetting the desired volume of AutoMACS Buffer into the top of the column and subsequently use the plunger provided to “push” all the remaining contents into a collection tube. The collection tube will now contain the positively selected target cell fraction.

Purification of the eluted positively selected target cell fraction

The eluted positively selected target cell fraction can now be further purified by subjected to a washing step to remove any impurities in the cell suspension. This is done by centrifuging the cell suspension and remove the supernatant leaving the highly purified target cell pellet at the bottom of the collection tube, allowing further downstream experimental procedures.

Similarly, the negatively selected fraction can also be purified using this method to obtain the cell pellet for downstream research procedures

Buffy Coat/Layer (Peripheral Blood Mononuclear Cell - PBMC)

Peripheral Blood Mononuclear Cell (PBMC)

A peripheral blood mononucleated cell (PBMC) is any WBC having a single round nucleus. Typically lymphocytes, monocytes and macrophages are grouped under this classification. These WBCs are a critical component in the immune system to tackle against infection and adapt to pathogenic intruders. The lymphocyte population consists of T cells (CD4 and CD8 positive ~75%), B cells and NK cells (~25% combined).

These cells can be extracted from whole blood using ficoll, a hydrophilic polysaccharide that separates layers of blood, which will separate the blood into a top layer of plasma, followed by a layer of PBMCs and a bottom fraction of polymorphonuclear cells (such as neutrophils, basophils and eosinophils) and erythrocytes. The polymorphonuclear cells can be further isolated by lysing the red blood cells. 

Buffy Layer/Coat

The buffy coat/layer is the fraction of an anti-coagulated blood sample that contains most of the Peripheral Mononuclear Blood cells (Lymphocytes and Monocytes) and platelets following density gradient centrifugation of the blood. 

After centrifugation, one can distinguish a layer of clear fluid (the plasma), a layer of red fluid containing most of the red blood cells, and a thin layer in between. Making up less than 1% of the total volume of the blood sample, the buffy coat (so-called because it is usually buff in hue), contains most of the white blood cells and platelets. The buffy coat is used mostly for research purposes, to extract DNA from the blood of. The RBCs are discarded most of the time as they have limited diagnostic or research value since they do not have nucleus that contain genetic material.