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Cell Biologics' products can be purchased through Fisher Scientic by searching Cell Biologics' product catalog number or key words...

  • Medium Promotion, Buy 3 Bottles, get the 4th Free on Feb. 2018.

    Promo Code: CB0218.

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AACR April 14-18, 2018 - Chicago

Experimental Biology April 22-24, 2018 - San Diego


  • Get Free Gelatin-Based Coating Solution per primary cell order,  which can be used for most primary cell culture from Cell Biologics through 2018.



Culture Primary Mouse Endothelial Cells

General Protocol for Recovering or Freezing Primary Cells

All cell culture procedures must be conducted in a bio-safety cabinet.

Any and all media, supplements, and reagents must be sterilized by filtration through a 0.2 µm filter.

Use aseptic technique to prevent microbial contamination.

Cryo-preserved cells must be stored in liquid nitrogen or seeded immediately upon arrival.


Review the information provided on the Cell Biologics website about appropriate culture media (e.g. serum and other supplements).

Use pre-warmed (37°C) cell culture media (30-50 ML) to recover cryo-preserved cells and when changing media or splitting cells.

Coating of flasks or dishes

Coat sterile culture dishes or flasks with Gelatin-Based Coating Solution (Cell Biologics, Catalog No. 6950) for 2 min and then aspirate the excess solution before seeding cells.

Handling of Arriving Live Cells

When you receive the live cells in a T25 or T75 flask, remove the sticker from the filter cap, and keep the flask with 6-20 ml existing medium in 37°C CO2 incubator for 1 hour before replacing the desired Cell Biologics' cell culture medium. Either split the 90% confluent cells from a T25 flask to a T75 flask after 1 hour or let the cells grow in the T25 flask with the desired Medium (such as M1168) for 12-48 hours before subculture.

The recommended split ratio for primary cells is 1:2.

Cell recovery from cryovial

·       Quickly thaw cells in cryo-vial by incubating them in a 37°C water bath for <1 min until there is just a small bit of ice left in the vial.

·       Promptly remove the vial and wipe it down with 70% ethanol.

·       Transfer cells from the vial to a sterile centrifuge tube. Add 8-10 ml of pre-warmed Cell Biologics Cell Culture Medium.

·       Flush the vial with an additional 0.5-1 ml of medium to ensure complete transfer of cells to the centrifuge tube.

·       Centrifuge cells at 200 g for 5 minutes.

·       Aspirate the supernatant and resuspend the cell pellet in 6 ml of Cell Biologics’ Cell Culture Growth Medium.

          Recovery cells from cryovial in 10% FBS for the first and second days.

·       Add resuspended cells into a T25 flask pre-coated with Gelatin-Based Coating Solution (Cell Biologics, Catalog No. 6950). It will take 3-5 days for cells to reach confluence.

·       Place the T25 flask in a humidified, 5%-CO2 incubator at 37°C.

·       Change culture media the following day to remove non-adherent cells and replenish nutrients.

·       Change cell culture medium every day when cells are >70% confluent.

·       Cells should be checked daily under a microscope to verify appropriate cell morphology.

Expansion of cultured primary cells

·       Remove and discard the cell culture media from the flask.

·       Flush the adherent layer 2 times using a 5 ml sterile pipette with sterile PBS (1X) without calcium and magnesium to dislodge loosely attached cells and remove fraction.

·       Remove and discard the wash solution from the flask.

·       Incubate cells with warm (37°C) 0.25% Trypsin-EDTA solution (Cell Biologics, Catalog No. 6914) for 3-5 minutes. Use 3.0 ml of Trypsin-EDTA solution when collecting cells from T75 flasks, and 2 ml when using T25 flasks. As soon as cells have detached (the flask may require a few firm gentle taps), add 8-10 ml of Cell Biologics’ Cell Culture Medium supplemented with 5-10 % FBS to a T25 or T75 flask (the FBS will neutralize the trypsin).

·       Plate cells in fresh flasks or plates precoated with Gelatin-Based Coating Solution in a humidified, 5%-CO2 incubator at 37°C.

·       Change culture media the following day to remove non-adherent cells and replenish nutrients.

·       Cells should be checked daily under a microscopy to verify appropriate cell morphology.

·       Change culture medium every 24-48 hours. Please note that the medium should be changed every day when cells are >70% confluent to remove non-adherent cells and replenish nutrients. Pre-wash cells with 1X PBS 1-2 times whenever replacing the medium.

·       We recommend splitting primary cells at the follow ratio

·       The recommended split ratio for primary murine cells is 1:2.

·       Confluent monolayer of primary cells grown in a T75 flask may be expanded on a 6-well plate ready for use in experiments under the cell culture conditions specified by Cell Biologics.

Primary cells differ from cell lines in many ways.  Here’s what you should know about primary cells:


  1.  Primary cells have a finite life span. They are exactly like the cells that you would find in your own body, so they do die after a certain period of time in culture. The amount of time primary cells survive in culture varies according to cell type.  
  2.  Primary cells can be diverse. Cancer cell lines often come from a single patient – for example, HeLa cells came from one woman, Henrietta Lacks. Primary cells, on the other hand, can come from a variety of people. Having a variety of primary cell types from many different donors is especially useful when carrying out early drug testing— it’s important to ensure that the drug is effective for everyone. Before testing the drug in human patients, researchers can use primary cells from different donors to verify that the same effects are observed. 
  3. Primary cells can change in culture.  For this reason, and because primary cells do not live forever, Lifeline Cell Technology strongly suggests carrying out experiments on primary cells using earlier passages.
  4. Primary cells have not been modified in any way. Except for the enzymatic and/or physical dissociation required for extracting the cells from their tissue of origin, primary cells are not altered in any way. Immortalized cell lines, on the other hand, are often transformed either with cancer genes, viruses or other inducible modifications, which could alter the outcome of experiments.
  5. Primary cells are finicky. Cell lines are hardy, mostly because they have to last a long time in culture, and be capable of surviving multiple rounds of cryopreservation and thawing. Primary cells, on the other hand, tend to be more difficult to maintain in culture.