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Cell-Ess® Serum Replacement Instructions

Cell-Ess – Cat. No. 25757-710-002, -003 and  -004

Please read before using Cell-Ess Serum Replacement:

  •  You must adapt your cells to Cell-Ess (see 2.2), if you are using a cell line
  • Cell-Ess is packaged as two components: Cell-Ess Base and Cell-Ess Growth Factor Cocktail (GFC).
    – Remember to add Cell-Ess Growth Factor Cocktail (GFC) before use. (See 1.1 below.)
  • Use Cell-Ess just like serum, replacing it ml-for-ml to start, then optimize.
    – If your current application requires less than 10% FBS contact us for optimized protocol.
  • Cell-Ess is chemically defined, serum-free and animal-free.
  • Shelf life prior to mixing is one year from date of manufacture. After mixing, 4-6 weeks.

Cell-Ess Product Performance Warnings:

  • Do not freeze – store at 2°-8°C and protect from light
  • Do not heat inactivate – this is unnecessary with a sterile, non-animal product
  • Do not filter.  Cell-Ess has a slightly cloudy appearance.  This is normal.
  • Use caution with antibiotics. (see 2.3)
  • Use extreme caution if using trypsin. (see 2.3)

Watch this “How To Use Cell-Ess” video before you start.

 

1.1 Addition of Growth Factor Cocktail

Important Note: Add the Growth Factor (GFC) to Cell-Ess base just prior to use. Once mixed, Cell-Ess will have full performance for 4 weeks. We recommend the good practice of noting the mixing date on the bottle used, both to mark the date and also to confirm that the Growth Factor Cocktail has been added.

Mixing directions:

Cell-Ess can be mixed directly in the Cell-Ess base bottle or in separate containers.  Add 1ml of Cell-Ess Base to each GFC vial. Swirl gently to mix.  Transfer the contents of the Growth Factor Cocktail (GFC) vial(s) into the Cell-Ess base and rinse the Growth Factor vial twice with Cell-Ess to ensure complete transfer of Growth Factor. Place the bottle on the hood surface and swirl vigorously without creating bubbles. After mixing, store for 4-6 weeks at 2°-8°C and avoid light.

 

Product Options and Matching GFC

GFC

Cell-Ess Base

2 GFC vials
Part 25767-711-011 prepares 50 ml of Cell-Ess at a time

100 ml Cell-Ess

1 GFC vial
Part 25767-711-012 prepares 500 ml of Cell-Ess

500 ml Cell-Ess

2 GFC vial
Part 25767-711-012 prepares 1000ml of Cell-Ess

1000 ml Cell-Ess


2.1 Using Cell-Ess with Primary Cells

Many primary (Ex-vivo) cells may be directly cultured in Cell-Ess and your base medium. Use Cell-Ess at the same concentration used with serum (typically 10%).

2.2 Using Cell-Ess with immortalized cell lines

As with all serum-free products, for best results and cell performance, first wean your immortalized cell lines away from FBS.

Sequential Adaptation:
These sequential adaptation steps are strongly recommended for weaning your cell lines from FBS to Cell-Ess.

Expect these adaptation steps to take several weeks. Do not rush this process.

We recommend using Cell-Ess at the same concentration that you use FBS (e.g. 10%).

Once adapted from FBS, then experiment with the Cell-Ess concentration to optimize.

Important note:
Cell-Ess has a 4-6 week shelf life after GFC addition. Some adaptations may take more than 6 weeks; therefore, it is highly recommended to make up new batches of Cell-Ess with growth factor as needed to last through the adaptation period.

Sequential Adaptation Steps

seqadaptsteps

mixdiradaptsteps


Notes and tips on adaptation steps:

  • We recommend that you first adapt your host cell line to Cell-Ess prior to conducting any other cell applications, cell function or expression assays.
  • If you have trouble getting your cells past any step change, go back and passage the cells 2-3 times in the previous media ratio.
  • We strongly recommend that you take these precautions during adaptation. Contact us for instructions to increase GFC concentration during later stages of weaning, or guidance on plating substrates or additives like HSA.
    • Make a frozen stock of the cells in the serum-supplemented medium prior to adaptation.
    • Keep a culture of cells at each adaptation step as a fallback in case there are problems with the next passage


2.3 Points to Consider in Serum-Free Culture

Overall, cells in serum-free culture are more sensitive to extremes of pH, temperature, osmolality, mechanical forces, and enzyme treatment. As a result, working without FBS can be challenging, but there are many important benefits.

Reagents and Supplements

There are a few functional components present in serum which may need to be reproduced in a serum-free medium.  Depending upon your application, you may need to add adherence factors, specific differentiation factors, etc. See the chart at 3.1.

Growth Factors

Cell-Ess includes a proprietary cocktail of synthetic growth factors designed to replace the function of FBS in most applications.  If you have special needs for growth factors or would like to discuss a customized Cell-Ess for your application, please contact us.

Passaging Cells- Avoid Trypsin

Avoid trypsin use when passaging cells. Trypsin use will signifi cantly impact cell adherence and cell health as FBS percentages are reduced during weaning steps. Trypsin is an animal product and alternatives are available to maintain animal component free culture. Call or email us at EssHelp@EssentialPharma.com for alternatives.

Antibiotics

It is best not to use antibiotics in serum-free media.  If you do, we recommend that you use 3 to 4-fold less than you used in a serum-supplemented medium.  This is because serum proteins bind antibiotic added. Reduced serum levels leaves more antibiotic available to be toxic to certain cells.

Seeding Cells at Higher Density

Cells must be in the mid-logarithmic phase of growth with viability above 90% prior to adaptation.  Seeding cultures at a higher density than normal at each passage during adaptation may help the process.  Because some percentage of cells may not survive in the new culture environment, having more cells present will increase the number of viable cells to further passage.

Clumping

Cell clumping often occurs during adaptation to serum-free medium.  We recommend that you gently triturate the clumps to break them up when passaging cells.

Morphology

It is not uncommon to see slight changes in cellular morphology during and after adaptation.  As long as doubling times and viability remain good, slight changes in morphology should not be a reason for concern.

Functionality

As with any significant change in your media, reagents or supplements, it is important to validate that your cells within your systems have comparable functionality when grown in Cell-Ess.


Figure 3.1   A Flow Chart of decisions required when using Cell-Ess

flowchartdecisions2Questions, comments, or need help?

For questions, comments, and tech support, please email us at EssHelp@EssentialPharma.com or phone us in the USA at 844-EssProd (844) 377-7763.