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PBMC Isolation Protocol

What is PBMC Isolation?

PBMC isolation is the process of separating mononuclear cells from whole blood.

There are two primary cell isolation techniques that are used to extract PBMC from the blow. These techniques include: Ficoll-Paque Density Centrifugation and SepMate Centrifugation

Ficoll-Paque Density Centrifugation

Ficoll-Paque is a long-chain molecule entity that is attracted to water and easily dissolves. It is used during the centrifugation process to aid in the separation of mononuclear cells from the buffy coat, plasma, and other components of blood.

When this method is employed, the labratorist will place the Ficoll-Paque at the bottom of the conical tube and then place the blood sample on top. Ficoll-Paque centrifugation takes more time than other forms of centrifugation, with the process lasting an hour or more in most cases.

SepMate Centrifugation

SepMate centrifugation differs from the Ficoll-Paque method, as it does not require laboratory technicians to layer the blood in a specific sequence when preparing the conical tubes. SepMate also eliminates the need for additional purification steps that are required when the Ficoll-Paque method is used. This results in a significant reduction in the amount of time required to successfully separate PBMCs from the blood, cutting total time needed down to approximately 30 minutes.

What are the Benefits of Primary Cell Isolation?

There are many reasons why healthcare professionals, researchers, and other experts choose to use PBMC isolation methods.

Some of the most compelling advantages of cell isolation include:

Increased Cell Viability

By using PBMC processing, researchers are able to secure a higher density of usable cells, reducing the number of samples they require per study or experiment. Additionally, these cell culture steps also make it easier to adequately cryopreserve PBMC samples for future use.

Biomarker Identification

When PBMCs are isolated from other cells, it is easier to study their specific traits and behaviors. PBMCs are routinely studied in hopes of discovering disease biomarkers that will contribute to more advanced treatments and cures.

Comparison Studies

Researchers often need both healthy and disease state PBMCs in order to conduct comparison studies. These studies provide a means of analyzing how healthy cells and unhealthy cells will react to a specific stimulus. Comparison studies are frequently used to determine the efficacy of pharmaceutical drugs, and are of paramount importance in regards to eliminating any risks when developing new treatments and medications.

Our Isolation and Preservation Services

At BuyPMBCs, we follow strict PBMC isolation protocols when developing our biomaterial products. We routinely isolate PBMCs and cell subsets using positive or negative selection methods that have a >90% average lot viability and purity post-thaw. With over 40 years of experience, we have performed hundreds of cell isolations from peripheral blood and have experience with effective recovery of cells from patients with a wide range of diseases.

We are also proficient in the safe storage of isolated PBMC products. We freeze cells using a controlled rate freezing method to minimize the formation of ice crystals, which can rupture the cells and compromise sample quality.

We also utilize cryoprotective agents such as a DMSO and FBS cocktail in the freezing media to further eliminate ice crystal formation. These reagents have a low toxicity and easily penetrate cell membranes, helping to stabilize osmotic pressure.

Ultimately, we provide only the most stable and viable PBMC products to our clients, and all samples are extensively characterized and available immediately from our large inventory of donors with varying demographic profiles.

How to Correctly Thaw Your Isolated PBMCs

Keep the cells on dry ice

When transferring the vials of cells from the freezer to your workstation, even if it’s a short distance, keep the vials on dry ice so that the cells stay cold. Additionally, don’t try to thaw more than four vials at a time. Focusing on more vials may divide your attention and prevent you from treating each vial with optimum care.

Warm the cell medium

Place the container of medium that you will be using to suspend the cells into a water bath that is warmed to 37°C ± 3°.

Warm the cells in a water bath

Hold your vials in a water bath that is warmed to 37°C ± 3°. Do not immerse the vial below the cap level, as this can allow water to leak inside and contaminate your samples. Keep a close eye on your vials; when there is still one ice crystal left on the external surface of the vial, remove the vial from the water bath. Wipe it down using a sterile alcohol wipe, especially around the cap, to prevent your cells from becoming contaminated.

Transfer the cells to a new tube

Prepare and label a 50 mL tube. (If you have a small aliquot, you may choose to use a 15 mL tube.) Pour the PBMCs from the cryo-vial into the new tube.

Add warmed cell medium

Measure 8 mL of the warmed cell medium. Slowly add it to the tube, swirling as you go to help the cells mix with the medium.

Rinse the original vial

To increase the amount of cells you recover, rinse the cryo-vial with 1 mL warm medium and pour it into the 50 mL tube.

Pellet the cells and discard the supernatant

Centrifuge the cells at 400xG for 10 minutes. If there is no pellet, centrifuge the cells again at 400xG for 15 minutes more. After centrifuging, discard the supernatant, as it contains the new cell medium mixed with the cryo-protective medium.

Add more cell medium and pellet the cells again

To ensure that all of the cryo-protective medium is removed from the cells, give the cells an extra rinse. To do so, add 10 mL more of the warmed cell medium and centrifuge the vial at 400xG for 10 minutes. Again, discard the supernatant when you are finished centrifuging and re-suspend the cells in whatever volume of the warm medium you need for your cell counting protocol. Be sure to mix (carefully) the cell solution to ensure that there are no clumps.

Count the cells

Use your laboratory’s preferred procedure to count the cells.

Rest the cells overnight

Before using the cells in cell-based assays, it is recommended that you rest the cells overnight at 37°C ± 3°.

There you have it—a 10-step method for thawing PBMCs. Precision for Medicine wants to help ensure you get the full value from your cryopreserved PBMCs. By following these procedures you’ll find that cells have a greater than 90% average lot viability and purity post-thaw.

How to Order

You can request a free quote online or give us a call at 1-855-222-5010.

 

Please note:

All of our products are intended for research or manufacturing use only unless otherwise arranged. Products are not to be used for human therapeutics, or for any other purposes, including: in vitro diagnostic purposes, in foods, drugs, medical devices or cosmetics for humans or animals, or commercial use.

 

About The Author

Florence Williams