Primary Human Mononuclear Cells Culture

These products are available in the US, Canada, and select European countries.

• Mononuclear Cell Culture System
• Mononuclear Cell Culture System Quality Control
• Protocol for Mononuclear Cell Culture
• Subcultivation Protocol for Mononuclear Cells
• Materials for Mononuclear Cell Culture

Human Mononuclear Cell Culture System

Mononuclear cells (MNC) represent the enriched lymphocyte and monocyte fraction of whole blood. They are isolated from umbilical cord blood and adult peripheral blood of healthy donors. Mononuclear cells from cord blood contain a relatively high percentage of primitive progenitor cells, while mononuclear cells from adult peripheral blood (hMNC-PB) contain a high proportion of mature immune cells.

Mononuclear Cells are produced from freshly collected whole blood, which is carefully separated by low-density gradient centrifugation, effectively removing the granulocytes. To avoid general cell damage, red blood cells are gently depleted by a proprietary protocol rather than using traditional osmotic lysis techniques. The resulting ultrapure Mononuclear Cells do not clump when thawed, and they exhibit superior viability and unaltered biological function. Immediately after isolation, the freshly prepared Mononuclear Cells are cryopreserved using proprietary serum-free freezing medium, Cryo-SFM. Each cryovial contains at least 25 million viable cells after thawing.

The Mononuclear Cell Medium is a highly nutritive short-term maintenance medium, which provides optimal cell viability for Mononuclear Cells from umbilical cord blood, adult peripheral blood, and other sources such as bone marrow. It is validated for maintenance of Mononuclear Cells for up to 48 hours before proceeding with your experiments.  The Mononuclear Cell Medium consists of a bottle of Basal Medium and one vial of SupplementMix. Combining the SupplementMix with the Basal Medium constitutes the complete Medium.

Mononuclear Cell Medium is recommended for

• Human Mononuclear Cells from Cord Blood (hMNC-CB)
• Human Mononuclear Cells from Peripheral Blood (hMNC-PB)

Mononuclear Cell Medium Supplementation Details

PromoCell^® Mononuclear Cell Medium contains all the growth factors and supplements necessary for optimal short-term maintenance of mononuclear cells. The Mononuclear Cell Medium does not contain antibiotics or antimycotics and is formulated for use in an incubator with an atmosphere of 5% CO₂.

Preparing Supplemented Media for Mononuclear Cells

• Thaw the Mononuclear Cell SupplementMix at 15 – 25 °C.
• Aseptically mix the supplement solutions by carefully pipetting up and down.
• Transfer the entire contents of each supplement to the Mononuclear Cell Basal Medium.
• Close the bottle and swirl gently to achieve a homogeneous mixture.

Storage and Stability for Mononuclear Cell Products

• Store the Mononuclear Cell Basal Medium at 4 – 8 °C in the dark and the Mononuclear Cell SupplementMix at -20 °C immediately after arrival. Do not freeze the Mononuclear Cell Basal Medium.
• If stored properly, the products are stable until the expiration date on the label.
• After adding the Mononuclear Cell SupplementMix to the Mononuclear Cell Basal Medium, the shelf life of the complete medium is 6 weeks at 4 – 8 °C.  Do not freeze the complete medium.
• For use, aliquot and prewarm only the volume of Mononuclear Cell Medium needed for single use, and keep the remaining medium refrigerated at 4 – 8 °C.
• After delivery, cryopreserved cells should be seeded immediately, or stored in liquid nitrogen.

Mononuclear Cell Culture System Quality Control

Quality Control for Mononuclear Cell Medium

All lots of Mononuclear Cell Medium are subjected to comprehensive quality control tests using primary human mononuclear cells from cord blood and adult peripheral blood. Each lot of Mononuclear Cell Medium is checked for short-term maintenance of unaltered function and viability of mononuclear cells. Approved in-house lots of Mononuclear Cell Media are used as a reference. In addition, all lots of media have been tested for the absence of microbial contaminants (fungi, bacteria, mycoplasma).

Quality Control for Human Mononuclear Cells

Rigid quality control tests are performed for each lot of Human Mononuclear Cells.

• Lots are routinely characterized by flow cytometry for viability and a series of cellular characteristics including cell size and granularity.
• Cells are tested for the absence of HIV-1, HIV-2, HBV, HCV, HTLV-1, HTLV-2 and microbial contaminants (fungi, bacteria, and mycoplasma).
• Detailed information is provided for each lot of Mononuclear Cells regarding the percentages of the major cellular sub-populations, such as lymphocytes, monocytes, and granulocytes.
• A detailed certificate of analysis (CoA) for each lot is available for download.

Human Mononuclear Cell Specifications

Product	Recommended Medium	Recommended Differentiation Media	Plating density
Human Mononuclear Cells from Peripheral Blood (hMNC-PB), single donor, ultra-pure	C-28030		1 x 106 cells per mL
Human Mononuclear Cells from Cord Blood (hMNC-CB), single donor, ultra-pure	C-28030 C-28021 C-39891	C-28020*	1 x 106 cells per mL

* Applicable for CFU-assays for hematopoietic progenitor cells (LTC-IC assays).

Protocol for Human Mononuclear Cell Culture

Important Notes:

• Upon arrival, seed cells immediately, or else store the cryopreserved cells in liquid nitrogen. Storage at -80 °C is not sufficient for cell preservation and causes irreversible cell damage.
• Handle using aseptic techniques in a laminar flow biosafety cabinet.

1. Prepare the medium
   Calculate the required culture surface area according to the recommended plating density (see table above) and the lot-specific cell number per vial stated on the certificate of analysis. Fill the appropriate volume of Mononuclear Cell Medium in cell culture vessels. Place the vessels in an incubator (37 °C, 5 % CO₂) for 30 minutes.
   
   
2. Thaw the cells
   Remove the cryovial from liquid nitrogen storage and immediately place it on dry ice— even for brief transportation. Under a laminar flow bench, briefly twist the cap a quarter turn to relieve pressure, then re-tighten. Immerse the vial into a water or thermal bath
   (37 °C) just up to the screw cap for 2 minutes. Ensure that no water enters the thread of the screw cap.
   
   
3.  Disinfect the vial and seed the cells
   Thoroughly rinse the cryovial with 70% ethanol under a laminar flow bench. Then, aspirate the excess ethanol from the thread area of the screw cap. Open the vial and transfer the cells with a 2 mL serological pipette (not a micropipette) to a cell culture vessel containing the pre-warmed medium from step 1, without resuspending.
   
   Note: Do not resuspend the cells at any time, as clumping may occur.
   
   
4. Incubate the cells
   Place the vessel in an incubator (37 °C, 5% CO₂). For complete recovery, leave the cells untouched for at least 18 hours. Do not disrupt the flask during this recovery stage. Change the medium after 18–24 hours.
   
   Note: Handling of the cells prior to complete recovery results in clumping.

Subcultivation Protocol for Mononuclear Cells

Important Notes:

• Use aseptic techniques in a laminar flow biosafety cabinet.

1. Harvest the cells
   Harvest the cell suspension and determine the cell number. Spin down the cells for 10 minutes at 240 x g.
   
   
2. Resuspend and reseed cells
   Discard the supernatant.  Add 1 mL of the Mononuclear Cell Medium and resuspend the cells by carefully pipetting up and down. Seed the cells according to the recommended seeding density in new cell culture vessels containing fresh medium prewarmed to 37 °C. Place the vessels in an incubator (37 °C, 5% CO₂).