CELL CULTURE MEDIA

BIOCHROM liquid, and powdered cell culture media are standardized according to the original formulation as recommended by the „ Tissue Culture Association" (Morton, H.J. In vitro 6, 89 [1970] and Morton, JJ. et al. In vitro 8, 106 [1972]). Quality of chemicals used meet in general the standards "pro analysi", and "Ph Eur", resp.

Any modifications from the original formulation are indicated on the label (e.g. w/o NaHCO₃).

Defined cell culture media gene-rally consist of four basic chemical groups: amino acids, carbohydrates, anorganic salts, and vitamins.

· Amino acids (both essential and non essential) are required for protein biosynthesis. Essential amino acids cannot be synthesized by the cell and must be supplied exogenously in the formulation. Non essential amino acids, on the other hand, depending on the metabolism of individual cells, may be synthesized by the cell and are not required in the formulation. However, a formulation that provides non essential amino acids may minimize the metabolic burden on the cell, thus allowing the cell to proliferate more rapidly or to produce a desired end-product more efficiently.

· Glucose is the most common carbohydrate used in mammalian cell culture. It provides the major energy or carbon source for biosynthesis. Through glycolysis, glucose is broken down to pyruvate, which is converted to essential metabolites and metabolic waste products in the citric acid cycle. Some media also contain sodium pyruvate as a carbon source. Galactose, which metabolizes to lactic acid at a slower rate is some-times substituted or used with glucose. This prevents excessive lactic acid accumulation and pH shift caused by the metabolic conversion of glucose to lactic acid.

· Inorganic salts are essential to cell growth and maintenance. They provide major ions in the form of sodium, magnesium, potassium, calcium, phosphate, chloride, sulphate and bicarbonate. Inorganic salts also help to maintain the cellular membrane by controlling the osmotic pressure. Additionally, they act as a buffer to protect cells from sharp pH fluctuations due to metabolite waste products.

· Vitamins are generally included in all formulations and function as catalysts or substrates to facilitate or control certain metabolic functions. Most cells require the B vitamins. Other vitamins or coenzymes may be required by some cells and are, therefore, included in some cell culture media,

· Most cell culture media contain phenol red as pH indicator that allows visual observation of pH change in the media due to cell metabolism or environmental factors. Other organic or inorganic components are often included in cell culture media to provide for specific nutritional or other requirements affecting cell growth. As serum-free media are being developed, an increasing number of components once contributed by serum are being replaced by chemically defined components.

POWDERED MEDIA

BIOCHROM instamed^® powdered media and salts are produced in specially designed particle reduction or blending equipment to control bioburden and the migration of inert materials into the product. Powdered media and salts are extremely hygroscopic and must be protected from atmospheric moisture; they are, therefore, packed into sealed glass (1 litre) or high density polyethylene plastic bottles (5 - 50 litres). Custom or larger package sizes are available on request, instamed^® powdered media should remain sealed and stored at +2 - +8°C in its original container.

Powdered media do not contain sodium bicarbonate. For addition use the following reference list:

Table 1

Medium	NaHCO₃ to be added 7.5% (w/v), cat.no. L1713, or L 1703, resp.
Medium	liquid solution ml/l	dry substance mg/l
Alpha medium, mod. MEM*	26.7	2000
BME Earle's	29.3	2200
Click-RPMI	15.7	1175
DMEM/Ham's F-12 (1:1)	32.5	2438
Dulbecco's MEM	49.3	3700
Glasgow MEM	36.7	2750
Grace's Insectcell medium	4.7	350
Ham's F-10	16.0	1200
Ham's F-12	15.7	1175
Iscove's	40.37	3024
Joklik MEM	26.7	2000
Medium 199 Earle's	29.3	2200
Medium 199 Hanks'	4.7	350
MEM Earle's*	29.3	2200
MEM Hanks'	4.7	350
MEM ,,spinner"	29.3	2200
octomed	28.0	2100
RPMI 1640	26.7	2000
Williams' medium E	29.3	2200

* HEPES can be used as a buffer (concentration: 20 mM) instead of sodium bicarbonate.

General preparation instructions for instamed^® -powdered media

· The volume of water (Cat.Nos. L0015, L0020) used should be 10% below final desired volume.

· While gently stirring the water, the entire content of powdered medium package should be dissolved. The appropriate amount of sodium bicarbonate (see resp. table) is then to be added.

· If necessary, pH is adjusted with either 1 N NaOH or 1 N HCI; the resulting pH should be 0.2 to 0.3 units below desired working pH.

· The final desired volume is then brought up, and the medium immediately sterilized by filtration.

· Ready-to-use media should be stored at +2-+8°C in the dark.

Preparation of (l0x)-liquid media

The dissolving technique is essentially the same (add sterile water at +20°- +37°C to powder medium up to volume, stir mixture to complete dissolution, sterilize by means of filtration). Please be aware that concentrated media preparations do not contain sodium bicarbonate.

Media that can be concentrated (l0x) with no problems:

Medium 199

Ham's F-10

Medium Ham's F-12 Medium mod.

Certain limitations apply for:

BME Basal Medium Eagle

MEM Minimum Essential Medium (Eagle)

Dulbecco's MEM mod.

RPMI 1640 Medium

(10x) concentrated solution is only possible with a pH value less than 2. Otherwise, (5x) or (2x) concentrations must be used instead, depending on the cystine/tyrosine concentration of the medium.

Concentrated solutions of Leibovitz Medium L-15 are not recommended.

LIQUID MEDIA

Equipment used io manufacture liquid products is designed specifically to prevent bioburden and endotoxin contamination as well as the migration of foreign materials (e.g. filter material, heavy metals or plasticizers) inio the product. Water used to manufacture liquid media, salts and reagents meet the criteria published in the Ph Eur for WFI ("Water For Injection"), processed by the Cyclodest technique, Liquid products are membrane sterilized, and aseptically dispensed into 100 ml or 500 ml glass bottles class 1/11. Larger packing sizes (5 - 500 litres) are available on request. All manufacturing processes and facilities are qualified and validated to ensure consistency and suitability for intended use. A Certificate of Analysis describing release criteria and actual test results is available on request for every single production lot. The majority of non-concentrated media products do contain sodium bicarbonate or HEPES, resp. Due to limited stability at +2 - +8°C, however, they do not contain L-glutamine, nor serum, nor antibiotics. Prior to use, L-glutamine from a stock solution (L-glutamine, 200 mM, Cat.Nos. K 0280 to K 0283) needs to be added to these media. The appropriate volumes of L-glutamine are:

Table2

Medium:	ml of L-glutamine, 200 mM, per litre medium
Alpha medium, mod. MEM*	10.0
BME Earle's / Hanks'	10.0
CMRL 1066	3.4
Coon's F-12	10.0
DMEM/Ham's F-12 (1:1)	12.5
Dulbecco's MEM	20.0
Glasgow MEM	20.0
Grace's Insectcell medium	20.5
Ham's F-10/Ham's F-12	5.0
Iscove's	20.0
Leibovitz L-15	10.3
McCoy's 5 A mod	7.5
MCDB 153	30.1
Medium 199 Earle's/Hanks'	3.4
MEM Earle's/Hanks'	10.0
octomed	3.0
PFEK 1	10.0
RPMI 1640	10.3
TC 100 insectcell medium	20.5
Williams' medium E	10.0

With identical volumes, a stock solution of N-acetyl-L-alanyl-L-glutamine (cat. no. K0202) can be used.

CONCENTRATED MEDIA

(l0x) concentrated liquid media do not contain sodium bicarbonate- NaHC0₃, (7.5% (w/v), cat. no. L1713) must be supplemented together with L-glutamine (200 mM, cat. no. K0280 to K0283) prior to use:

Table 3

L-glutamine (200 mM) and NaHCO₃, 7.5% (w/v) for 10x concentrated media

Medium	L-glutamine (200 mM) ml/l	NaHCO₃ (7.5%) ml/l
MEM Hanks'	10.0	4.7
MEM Earle's	10.0	29.3
Dulbecco's MEM	20.0	49.3
RPMl 1640	10.3	26.7

The ready-to-use medium is prepared by adding sterile water (cat. no. L0015) to 10 x concentrated medium up to the desired final volume.

To prepare one litre of MEM Earle's from concentrates (see also table 2), facultatively with non essential amino acids (NEA, cat. no. K0293), the following components are required:

MEM Earle's, (10x)	100 ml
L-glutamine, 200 mM	10 ml
NEA (if required)	10 ml
Sodium bicarbonate, 7.5%	29.3 ml
Antibiotics	10 ml
sterile water up to the final volume of	1000 ml

Instead of sodium bicarbonate, 20 ml/l of a 1 M HEPES buffer (cat. no. L1613) can be used.

The pH should be 7.2 to 7.4 at +37°C. Preparations from ten-fold concentrated media do have a pH of 6-8 (approx.). Media can be adjusted to the correct pH by using 1 N NaOH, and consecutively NaHCO₃. Sterilize immediately by membrane filtration, and dispense aseptically into sterile bottles. For maintenance of pH, cells should be grown in a controlled CO₂ atmosphere: e.g. NaHCO₃ at 2.2 g/l and pH 7.3 requires 7.0 % CO₂.

Stability of L-glutamine in liquid media

L-glutamine is not stable in dissolved form, if stored at +2 - + 8 C, or higher temperatures. Due to cyclisation of L-glutamine, toxic NH₃ is formed. Cell culture media containing L-glutamine and L-glutamine itself must, therefore, be stored at < - 20°C.

As a substitute for L-glutamine, which limits the shelf life of media, dipeptides containing L-glutamine can be used as a source. They are stable for extended time, even at room temperature. Cells can readily metabolize it into L-glutamine by cleaving the inherent peptide binding.

L-alanyl-L-glutamine (Ala-Gln) or glycyl-L-glutamine (Gly-Gln) are used in media, with the acetylated form of ac-Ala-Gln being more stable.

BIOCHROM offers the most common cell culture media in a version containing 'stable glutamine' (cat.nos. starting with FG ...).

Flavin in cell culture media

All commercially available cell culture media do contain 0.01 to 1.0 mg/l of flavines, due to their vitamin nature (in particular riboflavin, or vitamin B₂). However, flavines serve as potent photo sensibilisators, even if exposed to visible light. Absorbed light energy will be transferred by this sensibilisator to oxygen molecules. Unsaturated organic substrates may react with formation of toxic hyper- and peroxides, if exposed to this aggressive form of oxygen. To avoid this harmful effect on the media quality, media may be packed either in opaque material, not transparent even for visible light, or flavines have to be omitted from the media formulation. Necessary amounts may then be supplemented with the serum (at approx. 0.2 mg/1) or separately from a stock riboflavin solution.

CUSTOMIZED MEDIA

A customized media service is available with a minimum lot size for liquid media of 5 litres in either 100 ml, or 500 ml bottles. For powdered media, minimum lot size to guarantee homogeneous mixing of all components, is 100 litres. Packing sizes available range from 100 x 1 litre to 2 x 50 litres.

Delivery time is approx. 3 weeks for powdered media, and 5 weeks for liquid media from date of receipt of order. Please make use of the copy form, to avoid misunderstandings, and send us your formulation in writing.

Manufacturing customized media is a matter of confidence - trust our experience and take advantage of our service!

ALPHA MEDIUM – LIQUID AND POWDERED

Storage +2 - +8°C

Alpha Medium is a modified MEM medium, originally developed to grow Chinese Hamster kidney cells in vitro. Modification was done by an increase in the concentration of the amino acids, vitamins, lipoic acid, and pyruvate. Alpha medium supports the growth of bone mar-row cells under both monolayer and suspension culture conditions. The medium is also suitable for amniotic fluid cells in chromosome analysis.

Another widely used supplement modification are nucleosides.

Formulation (in mg/l)

References:

1. Stanners, C.P. et.al.; 1971; Nof.New.Biol. 230, 52

2. Stanners, C.P. et.al ; 1975; J. Gen. Virol. 29, 281

3. Earle, W.; 1943, J.Natl. Cancer Inst. 4, 165

NaCl	6800	Glycine	50
KCl	400	L-histidine•HCl•H₂O	41.9
NaH₂PO₄•2H₂O	158.3	L-isoleucine	52.5
MgSO₄•7H₂O	200	L-leucine	52.5
CaCl₂•2H₂O	264.9	L-lysine•HCl	73.06
D-glucose	1000	L-methionine	14.9
Phenol red	10	L-phenylalanine	33.02
NaHCO₃	2000	L-proline	40
Na-pyruvate	110	L-serine	25
		L-threonine	47.64
Adenosine	10	L-tryptophane	10.2
Cytidine	10	L-tyrosine	36.22
Deoxyadenosine	10	L-valine	46.9
Deoxycytidine	10
Deoxyguanosine	10	Ascorbic acid	50
Guanosine	10	Biotin	0.1
Thymidine	10	D-Ca-pantothenate	1
Uridine	10	Choline chloride	1
L-alanine	25	Folic acid	1
L-arginine•HCl	126.4	Myo-inositol	2
L-asparginine•H₂O	50	Lipoic acid	0.2
L-aspartic acid	30	Nicotinamide	1
L-cysteine•2HCl	31.3	Pyridoxal•HCl	1
L-cysteine•HCl•H₂O	100	Riboflavin	0.1
L-glutamic acid	75	Thiamine•HCl	1
L-glutamine	292	Vitamin B₁₂	1.36

Different from the original formulation, only 0.05 mg/l riboflavin are used due to risk of photo oxidative effects.

	Cat.No.	Unit
Alpha liquid medium	F0915	500 ml
with 2.0g/l NaHC0₃
without L-glutamine
with nucleosides

Alpha liquid medium	F0925	500 ml
with 2.0g/l NaHCO₃
without L-glutamine
without nucleosides

BME (BASAL MEDIUM EAGLE) – LIQUID AND POWDERED

Storage +2 - +8°C

BME and its modifications are widely used to support the growth of a broad spectrum of mammalian cells. The medium was originally designed as a chemically defined medium for the growth of mouse L cells and HeLa cells in a serum-deficient system. When used with a serum supplement, BME is useful for culturing many mammalian cell types, including normal and transformed cells.

Formulation (in mg/l)

Reference:

Eagle, H., Proc. Soc. Exp. Biol. Med. 89, 362 [1955]

	Earle's salts	Earle's diploid- salts	Hanks' salts
NaCl	6800	6800	8000
KCl	400	400	400
Na₂HPO₄•2H₂O			60
NaH₂PO₄•H₂O	140	140
KH₂PO₄			60
MgSO₄•7H₂O	200		200
MgCl₂•H₂O		200
CaCl₂	200	200	140
D-glucose	1000	1000	1000
Phenol red	10	10	10
NaHCO₃	2200	2200	350

L-arginine•HCl	21	Biotin	1
L-cystine	12	Folic acid	1
L-glutamine	292	Choline chloride	1
L-histidine	8	Nicotinamide	1
L-isoleucine	26	D-Ca-pantothenate	1
L-leucine	26	Pyridoxal•HCl	1
L-lysine•HCl	36.5	Thiamine•HCl	1
L-methionine	7.5	Riboflavin	0.1
L-phenylalanine	16,5	Myo-inositol	2
L-threonine	24
L-tryptophane	4
L-tyrosine	18
L-valine	23.5

Different from the original formulation, only 0.05 mg/l riboflavin are used due to risk of photo oxidative effects.

BME (BASAL MEDIUM EAGLE)

Preparation of (1x)-liquid media from concentrated media

	Cat. No.	(10x) Earle's salt sol.	(10x) Hanks' salt sol.
(10 x) Earle's saline	L1925	100	-
(10 x) Hanks' saline	L2025	-	100
(100 x) L-glutamine (200mM)	K0280/3	10	10
(100 x) Non essential amino acid	K0293	(10)	(10)
(7.5%) NaHCO₃	L1713*	29.3	4.7
bi-distilled, sterilized water ad 1000 ml

*NaHCO₃ (cat. no. L 1713), may be replaced by 20 ml of HEPES buffer (1M) (cat. no. L 1613) per litre of medium.

	Cat. No.	Unit
BME with Earle's salts	F0225	500 ml
with 2.2 g/l NaHCO₃
without L-glutamine

BME with Earle's salts (10x)	F0235	500 ml
without NaHCO₃
without L-glutamine

BME with Hanks' salts	F0245	500 ml
with 0.35 g/l NaHCO₃
without L-glutamine

CLICK - RPMI (1:1) - POWDERED

Storage +2 - +8°C

Formulation (in mg/I)

Reference:

Click et al., Cell. Immunol. 3, 264 [1972]

NaCl	7000	L-methionine	26.3
KCl	400	L-phenylalanine	47.5
Na₂HPO₄	425	L-proline	33
KH₂PO₄	30	L-serine	36
MgSO₄•7 H₂O	150	L-threonine	70
CaCl₂	70	L-tryptophane	15
Ca(NO₃)₂	34.8	L-lyrosine	55
D-glucose	1500	L-valine	67.5
Phenol Red	7.5
NaHCO₃	1175	Glutathione	0.5
		Biotin	0.1
L-alanine	17.8	Vitamin B₁₂	0.0025
L-arginine	100	D-Ca-pantothenate	1.125
L-arginine•HCl•H₂O	157.5	Choline chloride	2.5
L-aparagine	51.4	Folic acid	1.5
L-aspartic acid	36.6	Myo-inositol	19.5
L-cystine	55	Nicotinamide	1.5
L-glutamine	442	p-amino benzoic acid	0.5
L-glutamic acid	39.4	Pyridoxine•HCl	0.5
Glicine	20	Pyridoxal•HCl	1
L-histidine	7.5	Riboflavin	0.2
L-histidine•HCl•H₂O	55.3	Thiamine•HCl	1.5
L-hydroxyproline	10	Adenosine	12.5
L-isoleucine	90	Guanosine	12.5
L-leucine	90	Uridine	12.5
L-lysine•HCl	111.3	Cytosine	12.5

	Cat. No.	Unit
Click-RPMI powdered	T 125-01	1 l
with L-glutamine	T 125-05	5 l
without NaHCO₃	T 125-10	10 l
	T 125-50	50 l

CMRL 1066 MEDIUM

Storage +2 - +8 ⁰C

This protein free medium has higher levels of both nucleosides and vitamins, CMRL 1066 has been originally developed for clonal growth of monkey kidney cells and long term culture of L-cells. The medium has found utility for a range of cell lines originating from humans and monkeys. It will also support (in its slightly modified version CMRL 1415) non-transformed mouse cell lines.

Formulation (in mg/I)

Reference:

Parker, R.C. el al Special Publications, N.Y. Academy of Sciences, 5,303 [1957]

CaCl₂	200	Cholesterin	0.2
KCl	400	Choline chloride	0.5
MgSO₄•7H₂O	200	Folic acid	0.01
NaCl	6799	Myo-inositol	0.05
NaHCO₃	2200	Nicotinic acid	0.025
NaH₂PO4•H₂O	140	Nicotinic acid amide	0.025
		p-amino benzoic acid	0.05
L-alanine	25	Pyridoxal•HCI	0.025
L-arginine•HCl	70	Pyridoxine	0.025
L-aspartic acid	30	Riboflavin	0.01
L-cysteine•HCl•H₂O	260	Thiamine•HCl	0.01
L-cystine	20
Lglutamic acid	75	Cocarboxylase	1
L-glutamine	100	Coenzym A	2.5
Glycine	50	Deoxyadenosine	10
L-histidine•HCl•H₂O	20	Deoxycytidine	10
Hydroxyproline	10	Deoxyguanosine	10
L-isoleucine	20	Ethanol	16
L-leucine	60	NAD	7
L-lysine•HCl	70	FAD, disodium salt	1
L-methionine	15	D-glucose	1000
L-phenylalanine	25	Glutalhione	10
L-proline	40	5-methyl-deoxycytidine	0.1
L-serine	25	Phenol red	20
L-threonine	30	Na-acetate•3H₂O	83
L-tryptophane	10	Na-glucuronat•H₂O	4.2
L-tyrosine	40	Thymidine	10
L-valine	25	NADP, sodium salt •H₂O	1
		Polysorbate 80 VG	5
Ascorbic acid	50	UTP, trisodium salt•H₂O	1
Biotin	0.01
D-Ca-pantothenate	0.01

	Cat.No.	Unit
CMRL 1066 liquid	F0565	500 ml
with 2.2g/l NaHCO₃
without L-glutamine

COON's F-12 MEDIUM MODIFIED

Storage +2 - +8°C

Coon's medium was optimized to grow rat thyroid gland ceils, If compared to Ham's F-12, it has a twofold content of amino acids, pyruvate as well as ascorbic acid.

Formulation (in mg/I)

Reference:

Ambesi-Imbiobato, F.S., Parks, L.A.M. and Coon, H.G. Proc.Natl.Acad.Sci. USA 77,3455 [1980]

NaCl	7530	L-leucine	26.2
KCl	305	L-lysine•HCl	73
Na₂HPO₄•7H₂O	250	L-methionine	9
KH₂PO₄	68	L-phenylalanine	10
MgSO₄•7H₂O	104	L-proline	70
MgCl₂•6H₂O	106	L-serine	21
CaCl₂•2H₂O	165	L-threonine	23.8
CuSO₄•5H₂O	0.002	L-tryptophane	4
ZnSO₄•7H₂O	0.144	L-tyrosine	11
FeSO₄•7H₂O	0.8	L-valine	23.4
D-glucose	2000	Putrescine•2HCl	0.3
NaHCO₃	2500
Phenol red	1.25	Biotin	0.07
Na-pyruvate	220	D-Ca-pantothenate	0.5
		Nicotinic acid amide	0.04
L-alanine	18	Linoleic acid	0.09
L-asparagine•HCl	30	Pyridoxine•HCl	0.06
L-aspartic acid	26	Thiamine

POWDERED MEDIA

Preparation of (l0x)-liquid media

LIQUID MEDIA

Medium:

CONCENTRATED MEDIA

CUSTOMIZED MEDIA

ALPHA MEDIUM – LIQUID AND POWDERED

Unit

Earle's salts

Earle's diploid- salts

Hanks' salts

Unit

Unit

CMRL 1066 liquid