Freezing and Thawing FBS and Other Sera

Best practices for ensuring stability and performance of FBS and sera
How to freeze and thaw serum to safeguard its stability
Protect thawed FBS and other sera with safe refrigeration and storage

Best practices for ensuring stability and performance of FBS and sera

Animal serum is an ideal source of nutrients for cells in culture because it contains proteins, lipids, salts, vitamins, minerals, amino acids and other components necessary for growth. When stored and handled correctly, the performance characteristics of serum can be maintained for many years. Improperly storing and/or thawing serum products can decrease not only their immediate and long-term stability, but also their effectiveness.

Growth-promotion data demonstrate that serum maintains its growth-supporting characteristics throughout its shelf life when stored correctly. The table below contains stability data for Fetal Bovine Serum (FBS) for a five-year period. To assure stability, sera must be appropriately stored and handled.

Long-term Storage of SERA: Test Results

Multiple Passage is % of control growth, while Plating Efficiency (PE) and Cloning Efficiency (CE) are % PE and CE.

Product	Test	Year 0	Year 1	Year 2	Year 3	Year 4	Year 5
Irradiated FBS	Multiple Passage	110	104	116	126	93	90
	Plating Efficiency	61	36	51	35	40	50
	Cloning Efficiency	35	43	31	21	25	23
FBS	Multiple Passage	113	103	99	95	103	102
	Plating Efficiency	59	58	57	74	32	42
	Cloning Efficiency	49	56	42	47	28	30

How to freeze and thaw FBS and other serum to safeguard its stability

To efficaciously preserve the integrity of animal serum, it should be stored frozen and protected from light. The recommended storage temperature is -10 to -40 °C. At temperatures below -40 °C, storage bottles may become brittle, increasing the risk of breakage.

Multiple thaw/freeze cycles should be avoided as they will hasten the degradation of serum nutrients and can induce the formation of insoluble precipitates. For this reason, serum should never be stored in "frost-free" freezers. These appliances occasionally warm themselves to prevent internal ice deposits,and are therefore detrimental to the clarity and stability of frozen serum products.

Recommended Thawing Procedure for FBS and other sera

• Remove the serum bottles from the freezer and allow them to acclimate to room temperature for approximately 10 minutes.
• Place each container in a 30 to 37 °C water bath or incubator. Higher temperatures will degrade heat-labile nutrients. If using a water bath, do not submerge the bottles beyond the level of the serum content, to prevent the bottle caps from being submerged. Water baths are common sources of contamination for media and serum.
• Gently swirl or shake the bottles every 10 - 15 minutes until the serum is completely thawed.

Avoid cryoprecipitates when thawing serum (FBS)

The method used to thaw serum is vital to its optimum performance. The key to proper thawing is periodic agitation. If a bottle of serum is not regularly shaken or swirled as it thaws, salt and protein gradients will form throughout the liquid fraction. Within these gradients are high concentrations of salts, proteins, and lipids which can lead to the formation of crystalline or flocculent precipitates. These "cryoprecipitates" are not toxic to cell cultures, but they affect the appearance and consistent composition of the FBS or other animal sera.

Occasional, sparse cryoprecipitates are not uncommon, even in serum that is thawed using the recommended procedure. This is normal and will not affect product performance.

If serum is thawed incorrectly, increased cryoprecipitate may form, and is often insoluble. Filtering serum to remove cryoprecipitates is not recommended as it could result in the unintentional loss of nutrients, such as growth factors, mitogens, and other proteins.

Protect thawed FBS and other sera with safe refrigeration and storage

Use thawed serum promptly. Liquid FBS and other serum may be stored refrigerated (2 to 8 °C) up to four weeks. To avoid thaw/freeze cycles or discarding unused serum after more than four weeks of refrigeration, it is recommended that any unneeded serum be immediately dispensed into small, application-appropriate aliquots under sterile culture conditions, and refrozen to protect stability for future use.