JPH01157377A - Serum-free medium - Google Patents

Abstract

PURPOSE:To efficiently multiply and differentiate hepatic cells, etc., by preparing a serum-free medium containing an alkali metal chloride, alkaline earth metal chloride hydrate, alkali metal hydrogencarbonate, glycine, lysine or a salt thereof. CONSTITUTION:A serum-free medium containing 10-40mM at least one compound (component A) selected from the group consisting of pyruvic acid, aspartic acid, lactic acid, acetic acid, alpha-ketoglutaric acid, succinic acid, malic acid, fumaric acid and salts thereof as an essential component is prepared. Further the medium may be blended with 0.1-1mM at least one component (component B different from the component A) selected from the group consisting of aspartic acid, alanine, pyruvic acid, alpha-ketoglutaric acid, succinic acid, malic acid, fumaric acid and salts thereof besides the essential component. The serumfree medium contains about 1mM saccharide.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a serum-free medium. In particular, it relates to a serum-free medium for cell proliferation of hepatic parenchymal cells and maintenance culture of the differentiated cells.

[Prior art]

Tissue culture generally requires serum, especially fetal bovine serum. However, serum contains various physiologically active substances, including unknown substances. For this reason, when culturing tissues in a serum-containing medium, it is impossible to exclude the effects of these substances on the culture system, making it difficult to elucidate the mechanism of cell proliferation and cell physiology. In addition, it has problems such as serum heterogeneity, virus contamination, complicated culture medium preparation, high cost, and unsuitability for large-scale culture.

Furthermore, in serum-containing media, it is difficult to purify physiologically active substances produced and secreted by cultured cells, especially protein substances secreted extracellularly.

Therefore, the development of serum-free media has recently attracted attention.

It is known that in a living body, hepatocytes in a quiescent state (differentiated cells) (for example, hepatocytes remaining after partial liver resection) rapidly begin to proliferate and regenerate. That is, hepatocytes potentially have the ability to proliferate.

However, when culturing hepatocytes, a normal commercially available medium (
(contains no serum components), it is impossible to culture in a serum-free state, and even if serum is added, the hepatocytes will die in a short period of time.

[Problems to be Solved by the Invention] In view of the above-mentioned prior art, the present inventor has developed a medium in which hepatocytes, etc. can efficiently proliferate and differentiate without adding serum, and therefore can survive for a long period of time. That is, as a result of intensive research to prepare a serum-free medium in which cells can efficiently proliferate and differentiate, we have found that the desired purpose can be achieved by combining specific components, preferably in a specific composition ratio. They discovered this and completed the present invention.

[Means to solve the problem]

One feature of the present invention is that pyruvic acid, aspartic acid, lactic acid, acetic acid, α-ketoglutaric acid, succinic acid, malic acid, fumaric acid and their salts can be extracted from pyruvic acid, aspartic acid, lactic acid, acetic acid, α-ketoglutaric acid, succinic acid, malic acid, fumaric acid and their salts in a serum-free medium containing carbohydrates, inorganic salts, vitamins and amino acids. At least one selected one (hereinafter referred to as component A) at 10 to 40 mM
It is a serum-free medium characterized in that it contains a serum-free medium at a concentration of .

The essential components of the culture medium having the above characteristics of the present invention are as described above, and further include aspartic acid, alanine,
At least one selected from pyruvic acid, α-ketoglutaric acid, succinic acid, malic acid, fumaric acid, and their salts (referred to as component B. However, component A and component B are not the same kind) at a concentration of 0.1 to 1 mM. may be included.

Further, the serum-free medium having the above-mentioned first feature of the present invention preferably has the following composition, and contains about 1 mM of the second carbohydrate (+)M ii (eg, galactose, etc.). Note that glucose may not be substantially included.

■For inorganic salts, calcium ion is 0.01 to 0.
．． Contains about 1mM. Also, iron ions and zinc ions are required.

(2) Plasmin inhibitors (eg, aprotinin, ε-aminocaproic acid, etc.) can be added as other components.

Furthermore, addition of sphingomyelin is convenient for maintenance culture.

■As for amino acids, glutamine and thyronone may not be included.

The second feature of the present invention is a serum-free medium containing at least the following components: - Alkali metal chloride, alkaline earth metal chloride, hydrate, alkaline earth metal sulfate, hydrate, and diphosphate. Monosodium hydrogen, hydrate, alkali metal hydrogen carbonate, iron sulfate, hydrate, zinc sulfate, hydrate, arginine or its salts or ornithine or its salts, glycine, histidine salt, hydrate, isoleucine, leucine, Lysine or its salts, methionine, phenylalanine, proline, threonine, tryptophan, valine, biotin, pantothenic acid or its salts, choline chloride, folic acid, i-inositol, nicotinamide, pyridoxal or its salts, riboflavin, thiamine or its salts, pyruvin At least one alkali metal salt of an acid selected from lactic acid, acetic acid, α-ketoglutaric acid, succinic acid, malic acid, and fumaric acid and a plasmin inhibitor (e.g., aprotinin) The medium of the present invention further comprises the following components: It is preferable to incorporate at least one selected from: - copper sulfate - hydrate - selenite or its salt - manganese chloride - hydrate - asparagine - hydrate - aspartic acid or its salt - cystine or its salt Or cysteine or its salts, ascorbic acid, linoleic acid or its salts, riboic acid, menadione, sodium bisulfate adduct, vitamin B1
g - 1,25-dihydroxycholecalciferol - galactose - propionic acid or its salt - alpha-ketoglutaric acid or its salt - adenine or its salt - sphingomyelin, that is, the components contained in the serum-free medium of the present invention are inorganic salts. , amino acids, vitamins, and other components. Further, it is preferable that the serum-free medium of the present invention further contains hormones and the like. Note that protein components derived from serum are not substantially included.

(Inorganic salt) As the inorganic salt, the following components are preferably blended, preferably in the following composition ratio.

・Sodium chloride 7000-8000mg/
1. Potassium chloride 250-740 rng
/ 1. Calcium chloride dihydrate 10-30mg/
12.

・Magnesium sulfate heptahydrate 150-300 Tl1
g/E monosodium dihydrogen phosphate dihydrate 100
~300 mg/I.

・Sodium hydrogen carbonate 400-180 (1mg/
1. Iron sulfate heptahydrate 0.01~1.0■/
j2・Zinc sulfate・7hydrate 0.01~1.0■/
1 Furthermore, the following components are preferably blended in the following quantitative ratios.

・Copper sulfate pentahydrate 0.01~0.1■/l・
Selenite 0.0003-0.002 mg
/ 1. Manganese chloride tetrahydrate 0.001-0.0
5 ■/l Also, if necessary, metasilicate/9hydrate (especially sodium metasilicate/9hydrate) may be contained, preferably in an amount of 0.1 to 1.0 mg/42. can.

(Amino acids) As amino acids, the following components are preferably blended, preferably in the following composition ratios. Furthermore, the L-amino acid is preferably used.

・Arginine hydrochloride 20-200 mg/l or ornithine hydrochloride 10-100 mg/j!
・Glycine 20-80Ing/2・
Histidine hydrochloride/l hydrate 40~60■/2・isoleucine 50~60■/l・leucine
70-130■/l・Lysine hydrochloride
100-200■/l・Methionine
25z40■/l・Phenylalanine
30-40mg/l・Proline
50-200mg/ffi・Threonine
40-50mg/l tryptophan
15-50mg/i.

- Valine 65 to 75 mg/l As the amino acid, preferably, the following components are further blended, preferably in the following quantitative ratio.

・Asparagine monohydrate 10-20 ■/l・
Aspartate (sodium salt) 15-5000mg/ Tocystine dihydrochloride or cystine hydrochloride monohydrate
10-40 μ/2・Ornithine hydrochloride 10-170 mg/42 If necessary, serine, especially 10-50 μ/N, can also be contained.

(Vitamin) As vitamins, the following components are preferably blended, preferably in the following composition ratios.

・Biotin 0.01-0.2■/Calcium topantothenate 0.1-2■/1 ・Choline chloride 1-20■/l ・Folic acid
0.1~2■/l・i-inositol
# for 1-2!・Nicotinamide
0.1-2 mg/tibiridoxal hydrochloride
0.1-2 rng/l・Riboflavin 0.
01-0.2 ray(/A.thiamine hydrochloride
Further, as vitamins, the following components are preferably blended, preferably at the following V ratio.

・Ascorbic acid 10-100■/p.

・Linoleate (preferably sodium salt) 0.01~
0.1■/2, ribic acid 0.01-0.2 mg/
?・Menadione/sodium bisulfate adduct 0.001
~0.02mg/1 ・Active vitamin D 0.04-0.4 μg
/l Vitamin 812 0.01-1. Or
ag/E Here, as the linoleic acid salt, a type bound to albumin (particularly bovine albumin) is preferably used. In this case, it is preferable that albumin is bound at a ratio of 100 to 200 mg to 1 mg of linoleic acid.

(Others) As other components, the following components are preferably blended in the following composition ratios.

・Sodium pyruvate 10-4000mg/
Toabrotinin 100-300 μg/l (
or 1000 to 3000 units/l) As other components, the following components are preferably further blended, preferably in the following quantitative ratios.

・Galactose 100-200 mg/
Topropionate (preferably sodium salt) 25~
35mg//!・Alpha-ketoglutarate (preferably sodium salt) 8 to 25 mg/ffi ・Adenine hydrochloride 1 to 10 mg/f ・Sphingomyelin 0.05 to 0.1 mg/l (hormone) The medium of the present invention further contains a hormone. It is preferable to let The hormone preferably contains at least the following components, preferably in the following composition ratios.

・Insulin 10-100 u g/l
・Glucagon 1-10μg/l ・Triiodothyronine 5-50μg//2 ・Adrenocortical hormones (e.g. corticosterone hydrocortisone) 50-500ug/l Also, during proliferation culture, Evidermal Growth Factor 5-20μge
It is also possible to contain l.

Furthermore, the following components are preferably blended into the culture medium of the present invention in the following composition ratios as necessary.

・Kanamaynon 30-50mg/E・
Phenol red 30-50mg/F! In this specification, the alkali metal salts are preferably exemplified by sodium salts, potassium salts, etc., and the alkaline earth metal salts are preferably exemplified by calcium salts, magnesium salts, etc.

Examples of cells suitable for culturing in the medium of the present invention include hepatocytes. Hepatocytes are not particularly limited as long as they are derived from the mammary artery proboscis. Specific examples include humans, monkeys, dogs, rabbits, mice, and rats.

Methods for isolating hepatocytes include known methods such as perfusion method and collagenase treatment method.

Cultivation is performed by sequentially performing preculture, proliferation culture, and maintenance culture.

Preculture is a culture for adhering hepatocytes to a culture container.

As the preculture medium, the serum-free medium of the present invention (however,
may be free of hormones), fibrinogen (0.1-1%) and urokinase (10-5%) as needed.
0 units/mJ, fibronectin (1-2 μg/
ml) is used.

In addition, for cell attachment, the medium in which hepatocytes were cultured was added to 10%
May be added. The preculture conditions are around 37°C for about 1 to 5 hours.

In proliferation culture and maintenance culture, the composition and amount of hormones in the serum-free medium are changed depending on the purpose. The culture conditions are around 37°C [1 to 5 days (proliferation culture),
3 weeks or more (maintenance culture)], and the medium is replaced every 24 hours.

[Action/Effect] By using the serum-free medium of the present invention, proliferation of cells (especially hepatocytes) and survival and differentiation of hepatocytes during long-term culture, etc., which were impossible with conventional serum-free medium, can be achieved. will be held.

This medium also contains glucose, which is necessary for general cell growth.
Since it does not contain glutamine or tyrosine, the proliferation of general cells is inhibited, and it is easy to isolate and culture hepatocytes in particular.

As a result, the serum-free medium of the present invention is effective for cells, especially hepatocytes,
Especially culture of hepatic parenchymal cells (including proliferation culture and maintenance culture)
It is expected that this will be extremely useful.

In proliferation culture and maintenance culture, the composition and amount of hormones in the serum-free medium are changed depending on the purpose. The culture conditions are around 37°C [1 to 5 days (proliferation culture),
3 weeks or more (maintenance culture)], and the medium is replaced every 24 hours.

[Action/Effect]

By using the serum-free medium of the present invention, proliferation of cells (particularly hepatocytes) and survival and differentiation of hepatocytes during long-term culture, which were impossible with conventional serum-free media, are achieved.

Furthermore, since this medium does not contain glutamine, tyrosine, and glutamine, which are necessary for the growth of ordinary cells, the proliferation of ordinary cells is inhibited, and it is easy to separate and culture hepatocytes in particular.

As a result, the serum-free medium of the present invention is effective for cells, especially hepatocytes,
Especially culture of hepatic parenchymal cells (including proliferation culture and maintenance culture)
It is expected that this will be extremely useful.

〔Example〕

Examples will be given to explain the present invention in more detail, but the present invention is not limited thereto.

Example 1 A serum-free medium having the following composition was prepared.

(Inorganic salts) Sodium chloride 7600mg/Potassium monochloride 400/IV.

Calcium chloride dihydrate 15■/l Magnesium sulfate heptahydrate 250mg//! Monosodium dihydrogen phosphate dihydrate 150mg//! Sodium hydrogen carbonate 450mg/l iron sulfate
Heptahydrate 0.15■/l Zinc sulfate heptahydrate 0.15+ng/12.

Copper sulfate pentahydrate 0.02mg/j2 Selenite 0.002rng/ Manganese monochloride tetrahydrate 0.02mg/l Sodium metasilicate nonahydrate 0.25mg/ff1 (amino acid) L- Arginine hydrochloride 147mg/1
2.

L-asparagine monohydrate 15mg/ff1
L-aspartate sodium salt 60mg/IL-
Cystine dihydrochloride 15mg/l Glycine 75mg/42 L-Histidine hydrochloride lhydrate 52mg/12.

L-isoleucine 52mg/lL-
Leucine 105mg/1=L
-Lysine hydrochloride 145■/iL-Methionine 38n+g//! L-phenylalanine 33mg/j2L-proline 115mg/lL-threonine 60mg/12L-1-ributophane 30+ng/lL-valine
70■/iL-ornithine hydrochloride
17mg/ff1 (vitamin) Ascorbic acid 60■/l Biotin O, 1mg/lD-calcium pantothenate 1.0mg/f Cocoline chloride 10+ng/l folic acid
1.0mg/l linoleic acid sodium salt (bovine albumin binding type) 0.05mg/1
2.

i-inositol 10mg//! Riboic acid 0.1■/l Menadione/sodium bisulfate adduct 0.01■/i Nicotinamide 1. Omg/ff
i Viridoxal hydrochloride 1.0mg/j
2 Riboflavin 0.1■/P Thiamine hydrochloride 1.0mg/f Vitamin B +z O, 05mg, /
1 active vitamin D O, 2μg/R
(Others) Galactose 180■/f! .

Sodium pyruvate 3300mg//!
Sodium propionate salt 30/l Alpha monoketoglucuric acid sodium salt 20 mg/p.

Adenine hydrochloride 5 mg/l Sphingomyelin 0.05 mg/1 Aprotinin 2000 units/Q Example 2 (1) Isolation of hepatocytes ■ Rats weighing 200 to 300 g are used.

C a ” heated to 38°C to 40°C from the hepatic portal vein.
, Mg”, sodium pyruvate (550mg/
1), about 100 dill of Hank's solution containing HEPES (30mM, pH 7,4) was poured to thoroughly remove the blood in the liver, and then 0.025% collagenase (Si
H E P E S buffered Hanks solution supplemented with gma, Type 1.300 U/mg)
Perfuse 0ml.

■ Cut out the liver that has been softened by the above perfusion, cut it into small pieces,
Make a cell suspension by shaking gently with Hanks solution containing sodium pyruvate, centrifuge at low speed (l OXg) for 3 minutes,
Discard the supernatant. Hepatocytes are separated by repeating this 3 to 4 times. Add an appropriate amount of serum-free medium to make a hepatocyte suspension. The viability of hepatocytes is about 80-95% as determined by trypan blue.

(2) Culture method of hepatocytes Pour the required amount of serum-free medium into a flask (or petri dish) in advance, and add phipronectin (1 to 2 μg/
ml, add the hepatocyte suspension prepared thereto to the required final concentration, and start culturing at 37°C. After 3 to 4 hours, replace the medium with a new one, and then replace the medium every 24 hours.

Add various hormones depending on the purpose of the experiment. For example, when the purpose is proliferation, 3 to 5 mi of serum-free medium is placed in a flask with a bottom area of 30 cm1, and the cell density is adjusted to 3 x 10'/c+fl.

Hormones include insulin (50r+g/ml), glucagon (5ng/mR), triiodothyronine (5ng
Add corticosterone 50 ng/mn), corticosterone 50 ng/smoked or hydrocortiscin (100-200 ng/mf), and Evidermal Growth Factor (1 Ong/ml). The number of cells doubles on the 3rd to 4th day and begins to tightly fill the flask surface.

These proliferating hepatocytes secrete large quantities of albumin and other salivary proteins into the medium, and also retain well the liver's unique urea cycle enzymes. When maintaining and culturing hepatocytes for more than one week, remove Evidermal Growth Factor from the added hormones and add corticosterone (500n).
g/ml) or hydrocortisone (400-50
By increasing the amount added to 0 ng/mN), long-term culture becomes possible.

Example 3 In the composition of the serum-free medium of Example 1, sodium pyruvate 3300+ag/7! A serum-free medium was prepared in the same manner as in Example 1, except that sodium acetate (30mM) was replaced with 2460mg/l (30mM) of sodium acetate, and fresh mono-alanine was added to give a concentration of 89μ/P (1mM).

Example 4 (1) Isolation of hepatocytes According to Example 2 (1).

(2) Culture method of hepatocytes Pour the required amount of the serum-free medium of Example 1 into a flask (or petri dish) in advance, and add phipronectin (1 to 2
μg/mfl is added, the prepared hepatocyte suspension is added thereto to the required final concentration, and culture is started at 37°C. After 3 to 4 hours, replace the medium with a new one, and then incubate for 24 hours.
Replace the medium every hour.

Apply various hormones depending on the purpose of the experiment. For example, for the purpose of proliferation, the base area is 30c + J.
Add 3 to 5 ails of the serum-free medium of Example 1 to a flask so that the cell density is 3 x 10'/c++1.

Hormones include insulin (50ng/ml), glucagon (5ng/ml), and triiodothyronine (5ng/ml).
/af), corticosterone 501 g/ml or hydrocortiscin (100-200 ng/mβ),
Evidermal Growth Faku Ku (10ng/ml)
Add. The number of cells doubles on the 3rd to 4th day and begins to tightly fill the flask surface.

These proliferating hepatocytes secrete large amounts of albumin and other serum proteins into the medium, and also retain well the enzymes of the urea cycle unique to the liver. When maintaining and culturing hepatocytes for more than one week, switch to the serum-free medium of Example 3 and add insulin (50 ng/mQ), glucagon (5 ng/mff1), and triiodothyronine (5n) as hormones.
g/ml), corticosterone (500ng/In1
) or hydrocortiscin (400-500 ng/
ml) enables long-term culture.

Example 5 In the serum-free medium composition of Example 1, sodium pyruvate was added from 3300 mg/l (30 mM) to 110 mg/l (30 mM).
A serum-free medium was prepared in the same manner as in Example 1 except that sodium acetate was newly added to give a concentration of 2460 mg/l (30 mM). This medium is particularly useful as a maintenance medium for hepatocytes.

Example 6 The serum-free medium of Example 1 was prepared in the same manner as in Example 1 except that 3300 mg/l (30 mM) of sodium pyruvate was changed to 2240 mg/It (20 mM) of sodium L-lactate. Serum medium was obtained.

Example 7 The serum-free medium of Example 1 was prepared in accordance with Example 1 except that 3300 mg/l (30 mM) of sodium pyruvate was changed to 2700 mg/l (10 mM) of sodium corkate. A medium was obtained.

Claims (1)

[Scope of Claims] (1) Serum-free medium consisting of at least the following composition: - Alkali metal chloride - Alkaline earth metal chloride - hydrate - Alkaline earth metal sulfate - hydrate - Monosodium dihydrogen phosphate - Hydrate - Alkali metal hydrogen carbonate - Iron sulfate - Hydrate - Zinc sulfate - Hydrate - Arginine or its salt or ornithine or its salt - Glycine - Histidine salt - Hydrate - Isoleucine - Leucine - Lysine or its salt Salt, methionine, phenylalanine, proline, threonine, tryptophan, valine, biotin, pantothenic acid or its salt, choline chloride, folic acid, i-inositol, nicotinamide, pyridoxal or its salt, riboflavin, thiamine or its salt, pyruvic acid, lactic acid , at least one alkali metal salt of an acid selected from acetic acid, α-ketoglutaric acid, succinic acid, malic acid, and fumaric acid, and plasmin inhibitor (2), which further contains at least one selected from the following ingredients: Serum-free medium described in item (1), copper sulfate, hydrate, selenite or its salt, manganese chloride, hydrate, asparagine, hydrate, aspartic acid or its salt, cystine or its salt, or cysteine or its salts, ascorbic acid, linoleic acid or its salts, lipoic acid, menadione, sodium bisulfate adduct, vitamin B_1_2, 1,25-dihydroxycholecalciferol, galactose, propionic acid or its salts, alpha-ketoglutaric acid or its salts, Adenine or a salt thereof, sphingomyelin (3) The serum-free medium according to any one of claims (1) or (2), consisting of the following composition: alkali metal chloride, alkaline earth metal chloride, hydrate, alkali sulfate Earth metals, hydrates, monosodium dihydrogen phosphate, hydrates, alkali metal hydrogen carbonate, iron sulfate, hydrates, zinc sulfate, hydrates, copper sulfate, hydrates, selenite or its Salt, manganese chloride, hydrate, arginine or its salt or ornithine or its salt, asparagine, hydrate, aspartic acid or its salt, cystine or its salt or cysteine or its salt, glycine, histidine salt, hydrate, Isoleucine, leucine, lysine or its salts, methionine, phenylalanine, proline, threonine, tryptophan, valine, ascorbic acid, biotin, pantothenic acid or its salts, choline chloride, folic acid, linoleic acid or its salts, i-inositol, lipoic acid, Menadione, sodium bisulfate adduct, nicotinamide, pyridoxal or its salt, riboflavin, thiamine or its salt, vitamin B_1_2, 1,25-dihydroxycholecalciferol, galactose, pyruvate alkali metal salt, propionic acid or its salt, alpha Ketoglutaric acid or its salt, adenine or its salt, sphingomyelin, aprotinin (4) The serum-free medium according to claim (3), consisting of the following composition: sodium chloride, potassium chloride, calcium chloride, dihydrate, magnesium sulfate - Heptahydrate - Monosodium dihydrogen phosphate - Dihydrate - Sodium hydrogen carbonate - Iron sulfate - Heptahydrate - Zinc sulfate - Heptahydrate - Copper sulfate - Pentahydrate - Selenite - Manganese chloride, tetrahydrate, arginine hydrochloride or ornithine hydrochloride, asparagine, monohydrate, aspartate, cystine, dihydrochloride or cysteine hydrochloride monohydrate, glycine, histidine hydrochloride, monohydrate Isoleucine, leucine, lysine hydrochloride, methionine, phenylalanine, proline, threonine, tryptophan, valine, ascorbic acid, biotin, calcium pantothenate, choline chloride, folic acid, linoleic acid or its salts, i-inositol, lipoic acid, menadione - Sodium bisulfate adduct - Nicotinamide - Pyridoxal hydrochloride - Riboflavin - Thiamine hydrochloride - Vitamin B_1_2 - 1,25-dihydroxycholecalciferol - Galactose - Sodium pyruvate - Propionate - Alpha-ketoglutarate - Adenine hydrochloride - Serum-free medium according to claim (3), consisting of the following composition of sphingomyelin and aprotinin (5) - Sodium chloride 7000-8000 mg/l - Potassium chloride 250-740 mg/l - Calcium chloride dihydrate 10-30 mg /l Magnesium sulfate heptahydrate 150-300mg/l Monosodium dihydrogen phosphate dihydrate 100-300mg/l Sodium hydrogen carbonate 400-1800mg/l Iron sulfate heptahydrate 0 .1 to 1.0 mg/l, zinc sulfate, heptahydrate 0.1 to 1.0 mg/l, copper sulfate, pentahydrate 0.0
1~0.1mg/l・Selenite 0.0003~0.0
02mg/l・Manganese chloride・tetrahydrate 0.01~0.
05 mg/l・Arginine hydrochloride 20-200 mg/l or ornithine hydrochloride 10-170 mg/l・Asparagine monohydrate 0-20 mg/l・Aspartate 15-5000 mg/l・Cystine dihydrochloride 10-10 mg/l 2
0mg/l or cysteine hydrochloride monohydrate 20-40
mg/l ・Glycine 20-80 mg/l ・Histidine hydrochloride monohydrate 40-60 mg/l ・Isoleucine 50-60 mg/l ・Leucine 70-130 mg/l ・Lysine hydrochloride 100-200 mg/l ・Methionine 25 ~40mg/l ・Phenylalanine 30-40mg/l ・Proline 50-200mg/l ・Threonine 40-50mg/l ・Tryptophan 15-50mg/l ・Valine 65-75mg/l ・Ascorbic acid 10-100mg/l ・Biotin 0 .01-0.2 mg/l ・Calcium pantothenate 0.1-2 mg/l ・Choline chloride 1-20 mg/l ・Folic acid 0.1-2 mg/l ・Linoleic acid or its salt 0.01-0.2 mg/l l・
i-inositol 1-20mg/l ・Lipoic acid 0.01-0.2mg/l ・Menadione/sodium bisulfate adduct 0.001-0.02mg/l ・Nicotinamide 0.1-2mg/l ・Pyridoxal hydrochloride Salt 0.1-2mg/l・Riboflavin 0.01-0.2mg/l・Thiamin hydrochloride 0.1-2mg/l・Vitamin B_1_20.01-0.2mg/l・1・
25-dihydroxycholecalciferol 0.04~
0.4μg/l ・Galactose 100-500mg/l ・Sodium pyruvate 1000-4000mg/l
Propionate 25-35 mg/l ・Alpha-ketoglutarate 8-25 mg/l ・Adenine hydrochloride 1-10 mg/l ・Sphingomyelin 0.05-0.1 mg/l ・Aprotinin 100-300 μg/l (6) Further The serum-free medium according to any one of claims (1) to (5), containing 10 to 50 mg/l of serine. (7) The serum-free medium according to any one of claims (1) to (6), further containing at least one hormone selected from insulin, glucagon, triiodothyronine, and adrenocortical hormone. (8) The serum-free medium according to claim (7), containing 10 to 100 μg/l of insulin, 1 to 10 μg/l of glucagon, 5 to 50 μg/l of triiodothyronine, and 50 to 500 μg/l of adrenocortical hormone. (9) The serum-free medium according to any one of claims (1) to (8), which is substantially free of glucose, glutamine, and tyrosine. (10) Claims used for culturing hepatic parenchymal cells (
The serum-free medium according to any one of 1) to (9).