WO1994023017A1 - Composition utile en tant qu'additif d'un milieu de culture cellulaire, comprenant du colostrum et du serum - Google Patents

Composition utile en tant qu'additif d'un milieu de culture cellulaire, comprenant du colostrum et du serum Download PDF

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Publication number
WO1994023017A1
WO1994023017A1 PCT/FI1994/000089 FI9400089W WO9423017A1 WO 1994023017 A1 WO1994023017 A1 WO 1994023017A1 FI 9400089 W FI9400089 W FI 9400089W WO 9423017 A1 WO9423017 A1 WO 9423017A1
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WIPO (PCT)
Prior art keywords
culture medium
cells
colostrum
composition
less
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Application number
PCT/FI1994/000089
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English (en)
Inventor
Raimo Antero Pakkanen
Jouni Uolevi Aalto
Harry Gösta JALONEN
Ari Perttu Tapani Kanttinen
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Viable Bioproducts Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Viable Bioproducts Ltd. filed Critical Viable Bioproducts Ltd.
Priority to AU62091/94A priority Critical patent/AU6209194A/en
Priority to EP94909138A priority patent/EP0692023A1/fr
Publication of WO1994023017A1 publication Critical patent/WO1994023017A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/70—Undefined extracts
    • C12N2500/80—Undefined extracts from animals
    • C12N2500/84—Undefined extracts from animals from mammals

Definitions

  • composition useful as additive to a cell culture medium comprising colostrum and serum
  • This invention relates to a novel composition useful as additive to culture media for the cultivation of adherent cells.
  • the invention concerns further novel culture media and their use in the cultivation of such cells.
  • a basal medium In the cultivation of animal cells, a basal medium is traditionally supplemented with mammal blood serum.
  • mammal blood serum contains several partly unknown cell growth promoting agents, such as growth factors, vitamins, trace elements, hormones, binding proteins and attachment factors.
  • a serum suitable for most purposes is fetal bovine serum (FBS). The price of this serum has increased greatly in recent years because of the increasing demand and the limited availability of sufficiently pure sera.
  • FBS can be replaced with newborn calf serum, calf serum or even adult bovine serum.
  • FBS is, however, most effective. This is probably due to its high growth factor content.
  • Other important proteins in serum are attachment factors. These include, for example, fibronectin.
  • Most adherent cell types require additional attachment factors in cell culture medium although some cells can also synthezise them by themselves.
  • the objective of growing animal cells is the production, purification and characterization of secreted compounds such as monoclonal antibodies
  • the use of serum-free medium containing reduced amounts of protein and immunoglobulins makes downstream processing more simple and can greatly improve end-product purification and recovery.
  • bovine colostrum fraction useful as serum substitute for the cultivation of mouse hybridomas for production of monoclonal antibodies. Fractions of bovine colostrum were prepared and their ability to support the growth of mouse-mouse hybridomas in culture was tested. Whey was prepared from defatted colostrum by removal of casein using acid precipitation. An ultrafiltrate was obtained from cleared whey by filtration through membranes with a nominal molecular weight cut-off of 100000 Da.
  • the colostrum ultrafiltrate (colostrum fraction) so obtained contained 1.16 g/1 protein, 0.24 g/1 im unoglobulin G (IgG) and less than 0.24 EU (endotoxin unit)/ml endotoxins.
  • the effect of defatted colostrum, whey and ultrafiltrate as serum substitutes was studied by cultivation of hybridoma cells in minimal essential medium containing different concentrations of supplements. Under optimal conditions in ultrafiltrate-supplemented medium, the maximal cell concentration was 35 - 40 % of that obtained using 10 % fetal bovine serum, and antibody production per cell was equal to that achieved using fetal bovine serum.
  • the results of this study showed that the bovine colostrum ultrafiltrate provides a very attractive alternative to fetal bovine serum for production of monoclonal antibodies.
  • adherent cells An important group of cells used for the production of biochemicals are classified as adherent cells. Animal cells can be divided into two classes according to the manner in which they grow: suspension cells, which grow in suspended form, and adherent cells, which grow attached to a surface. Hybrido a cells are typically suspension cells while most of the other cells could be classified as adherent cells. Typical examples of adherent cells are e.g. Vero (kidney cells of the African Green Monkey), CHO-K1 (ovary cell line of the Chinese hamster) and 3T3-fibroblast. The Vero and CHO-K1-cells are widely used in the biotechnical industry. Vero cells are used in the production of viruses and viral proteins and CHO-K1- and CHO-cells are often used as host cells in large scale production of recombinant proteins.
  • adherent cells e.g. Vero (kidney cells of the African Green Monkey), CHO-K1 (ovary cell line of the Chinese hamster) and
  • Bovine colostrum resembles FBS in the respect that it contains growth factors.
  • the growth factor content of colostrum is high.
  • a colostrum ultrafiltrate could be used to replace FBS in hybridoma cell cultures.
  • colostrum does not contain enough attachment factors for many adherent cells. Such attachment factors can be found in FBS as well as in bovine serum (BS).
  • colostrum ultrafiltrate fraction The effect of the colostrum ultrafiltrate fraction on the growth of adherent cells has also been tested. Vero and CHO-K1 cells were grown in a basal medium containing varying amounts of colostrum ultrfiltrate according to the method described in the paper by R Pakkanen et al. A minor effect could be observed at colostrum ultrafiltrate concentrations ranging from 10 to 15 %, but the effect was very small compared to that caused to the growth of the hybridoma cells.
  • bovine serum contains the important attachment factor fibronectin.
  • fibronectin added to colostrum possesses an advantageous effect on the proliferation of adherent cells.
  • the addition of fibronectin to a colostrum fraction according to this invention did not affect the growth of adherent cells.
  • Another object of the invention is to provide an efficient culture medium for the cultivation of such cells.
  • Yet another object of the invention is to provide a method for the growth of adherent cells, by utilizing the culture medium of the invention.
  • the Figures IA to 6 describes the number of viable cells versus time during continuous cultivation on a culture medium comprising different attachment factor additive compositions and different amounts thereof.
  • the basal media used were DMEM for Vero cells and DMEM- F12 (1:1) for CHO-K1 cells supplemented with glutamine (4 mM), penicillin (100 units/ml) and streptomycin (100 ⁇ g/ml).
  • human transferrin (hTF) was added to the culture medium to give a concentration of 5 mg/1. All the percentages below are vol- %. For further details reference is made to the experiments.
  • Figure IA shows the number of viable CHO-Kl-cells versus time in a culture medium supplemented with 10 % FBS (•) and 13 % bovine colostrum ultrafiltrate (in the following abbreviated UF) (0).
  • Figure IB shows the number of viable Vero-cells versus time in a culture medium supplemented with 10 % FBS (•) and 13 % colostrum ultrafiltrate (in the following abbreviated UF) (0).
  • Figure 2A shows the number of viable CHO-Kl-cells versus time in a culture medium supplemented with 2 % BS (bovine serum) + 13 % UF (X)
  • Figure 2B shows the number of viable Vero-cells versus time in a culture medium supplemented with
  • Figure 3A shows the number of viable CHO-Kl-cells versus time in a culture medium supplemented with 1 % BS + 13 % UF (0) 1 % BS + 10 % UF (v) 1 % BS + 6.67 % UF ( ⁇ ) 1 % BS + 3.3 % UF (A)
  • Figure 3B shows the number of viable Vero-cells versus time in a culture medium supplemented with 1 % BS + 13 % UF (•) 1 % BS + 10 % UF ( ) 1 % BS + 6.7 % UF ( ⁇ ) 1 % BS + 3.3 % UF (_4)
  • Figure 4A shows the number of viable CHO-Kl-cells versus time in a culture medium supplemented with
  • Figure 4B shows the number of viable Vero-cells versus time in a culture medium supplemented with 0.5 % BS, no hTF added (t)
  • Figure 5A shows the number of viable CHO-Kl-cells versus time in a culture medium supplemented with 0.5 % FBS, no hTF added (•) 1.0 % FBS, " ( ⁇ ) 2.0 % FBS, no hTH added ( B ) 10 % FBS, no hTF added (_4) 1.0 % BS + 6.7 % UF, 5 mg/1 hTF (0) 0.33 % BS + 6.7 % UF, 5 mg/1 hTF (V) 0.60 % BS + 6.7 % UF, 5 mg/1 hTF (D) 1.0 % BS + 10 % UF, 5 mg/1 hTF ( .) no supplement of BS, UF or hTF added (X)
  • Figure 5B shows the number of viable Vero-cells versus time in a culture medium supplemented with 0.5 % FBS, no hTF added (•)
  • Figure 6 shows the number of viable CHO-Kl-cells versus time in a culture medium supplemented with 10 % FBS (•) 10 % UF (0) 10 % UF + 65 ⁇ g/ml of fibronectin (D)
  • a composition comprising a colostrum fraction, e.g. the colostrum ultrafiltrate as described in the experiments, and small amounts of serum efficiently enhances the growth of adherent cells when added to the culture medium for said cells.
  • the results obtained clearly exhibit a synergistic effect obtained by combining the two components, i.e. UF and BS. Comparative tests with the addition to the culture medium of plain BS, plain UF and various combinations of UF and BS show that the combinations give an essentially stronger effect on the growth of the adherent cell than what would be expected by adding the effects of the individual components UF and BS.
  • the invention thus provides a composition useful as additive to culture media for the cultivation of adherent cells, said composition comprising
  • colostrum fraction prepared by subjecting colostrum, from which fat and cellular debris have been removed by conventional methods, and from which colostrum optionally also casein has been removed by precipitation, to ultrafiltration by using a membrane having a cut off of 100,000 Da and recovering the filtrate, and
  • a serum component in an amount sufficient to enhance the proliferation of cells after the addition of a suitable amount of said composititon to a culture medium.
  • the invention further provides a culture medium useful for the cultivation of adherent cells, said culture medium comprising
  • colostrum fraction prepared by subjecting colostrum, from which fat and cellular debris have been removed by conventional methods, and from which colostrum optionally also casein has been removed by precipitation, to ultrafiltration by using a membrane having a cut off of 100,000 Da and recovering the filtrate,
  • the invention further provides a method of promoting proliferation of adherent cells in a culture medium said method comprising contacting an amount of cells with a culture medium according to the invention for a certain period of time.
  • the transferrin present in the serum may be enough.
  • the culture medium must be supplemented by additional transferrin.
  • the colostrum fraction is for practical reasons preferably obtained from bovine colostrum. Colostrum from other sources is, however, also believed to be useful for the preparation of the colostrum fraction to be used in this inventio .
  • the serum component is preferably bovine serum, but other sera such as horse sera are also believed to be effective.
  • the colostrum fraction has an endotoxin content of less than 5 EU/ l (units per ml), a total protein content of less than 4.5 mg/ml and an immunoglobulin content of less than 1.2 mg/ml.
  • compositions of the invention wherein the colostrum fraction has an endotoxin content of 1.0 EU/ml or less, a total protein content of 2.0 mg/ml or less and an immunoglobulin content of 0.25 mg/ml or less.
  • Such a fraction can be obtained by e.g. by subjecting the defatted colostrum to casein precipitation before the ultrafiltration.
  • composition of the invention comprises preferably serum component in an amount of at least 0.5 % of said composition.
  • composition according to the invention can further comprise one or more supplemental agents selected from the group comprising sodium selenite, ethanolamine, ⁇ - mercaptoethanol, bovine serum albumin and transferrin.
  • the culture medium of this invention can be prepared by adding separately appropriate amounts of colostrum fraction and serum to the basal growth medium
  • the culture medium is preferably made by adding to the basal growth medium an appropriate amount of a composition redily including the colostrum fraction and serum component.
  • the composition further includes added amounts of transferrin and optionally also other supplemental agents.
  • the culture medium has a concentration of the colostrum fraction ranging from about 1 % to about 20 % and a serum concentration that is at least 0.1 % of said culture medium.
  • the culture medium has a concentration of the colostrum fraction ranging from about 5 to 15 % of said culture medium.
  • adherent cells such as CH0-K1 (Chinese hamster ovary cells) and Vero (African green monkey kidney cells) as these two represent the technically most important adherent cells.
  • adherent cells such as CH0-K1 (Chinese hamster ovary cells) and Vero (African green monkey kidney cells) as these two represent the technically most important adherent cells.
  • BS bovine serum
  • UF colostrum ultrafiltrate
  • Chinese hamster ovary cells (CHO-K1, ATCC CCL 61) and African green monkey kidney cells (Vero, ATCC CCL 81) were obtained from Flow (Flow Laboratories, Rickmansworth, England) .
  • DMEM Dulbecco's Modified Eagles Essential Medium
  • DMEM-F12 (1:1) DMEM-F12 (1:1) (GIBCO) (for CHO-K1 cells) supplemented with glutamine (4 mM), penicillin (100 units/ml) and streptomycin (100 ⁇ g/ml).
  • GEBCO DMEM-F12 (1:1)
  • Stock cultures were maintained in 75-cm 2 plastic flasks (Cos ar, Cambridge, Mass, USA) supplemented with 10 % FBS (HyClone, Logan, Utah, USA).
  • BS bovine serum
  • FBS fetal bovine serum
  • UF colostrum ultrafiltrate
  • SIGMA human (holo)transferrin
  • the culture supernatants were removed and 150 ⁇ l 2xTE was added to each well. The plates were incubated at 37°C for about 10 min. Then, the culture supernatants were added back to the corresponding wells and the cells were suspended by pipetting the cultures. Cell counts were done in a haemocytometer using trypan blue exclusion to determine viability. Each counting point represents the average cell concentration of duplicate wells, and each well was counted only once.
  • FBS CH0-K1 cells tended to form large clusters and only a small cell population was spread.
  • Maximum growth of Vero cells was obtained in 13 % UF supplemented with 1-2 % BS, whereas no significant differences could be observed in CHO-K1 cultures supplemented with 0.6-2 % BS.
  • UF and BS Vero and CH0-K1 cells were cultured in 1 % BS supplemented with 3.3- 13 % UF and transferrin 5 mg/1.
  • CHO- Kl cells achieved maximum cell density in 6.7 % UF.
  • FIG. 5 shows the number of viable CHO-Kl-cells versus time in a culture medium supplemented with 10 % FBS, 10 % UF and 10 % UF + 65 ⁇ g/ml of fibronectin. As can be seen from the Figure, fibronectin added to the UF did not have any effect on the cell growth.
  • the colostrum fraction from which casein not has been removed before ultrafiltration which fraction also is useful in the composition and culture medium of the invention, is prepared according to the method described in the paper by R Pakkanen et al. referred to above with the only difference that casein is not precipitated before ultrafiltration.

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Abstract

Composition utile en tant qu'additif d'un milieu de culture destiné à la culture de cellules adhérentes, qui comprend une part de colostrum et du sérum en quantité suffisante pour favoriser la prolifération cellulaire après ajout d'une quantité appropriée de ladite composition à un milieu de culture. La présente invention concerne en outre des milieux de culture comportant une part de colostrum et du sérum, ainsi que l'utilisation desdits milieux de culture pour la culture de cellules adhérentes.
PCT/FI1994/000089 1993-04-01 1994-03-11 Composition utile en tant qu'additif d'un milieu de culture cellulaire, comprenant du colostrum et du serum WO1994023017A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU62091/94A AU6209194A (en) 1993-04-01 1994-03-11 A composition useful as additive to a cell culture medium, comprising colostrum and serum
EP94909138A EP0692023A1 (fr) 1993-04-01 1994-03-11 Composition utile en tant qu'additif d'un milieu de culture cellulaire, comprenant du colostrum et du serum

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US4165893A 1993-04-01 1993-04-01
US08/041,658 1993-04-01

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WO1994023017A1 true WO1994023017A1 (fr) 1994-10-13

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19619990A1 (de) * 1996-05-17 1997-11-20 Charlotte Adler Verfahren zur Herstellung von Kolostralmilchprodukten sowie deren Verwendung

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4440860A (en) * 1980-01-18 1984-04-03 The Children's Medical Center Corporation Stimulating cell growth
WO1992000014A1 (fr) * 1990-06-28 1992-01-09 Clar (Sarl) Procede de traitement du colostrum par chromatographie d'adsorption sur hydroxyapatite, fraction active de colostrum obtenue et milieu cellulaire contenant ladite fraction active
WO1993008264A1 (fr) * 1991-10-17 1993-04-29 Valio Bioproducts Ltd. Fraction du colostrum, sa preparation et son utilisation comme additif de bouillon de culture de cellules

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4440860A (en) * 1980-01-18 1984-04-03 The Children's Medical Center Corporation Stimulating cell growth
WO1992000014A1 (fr) * 1990-06-28 1992-01-09 Clar (Sarl) Procede de traitement du colostrum par chromatographie d'adsorption sur hydroxyapatite, fraction active de colostrum obtenue et milieu cellulaire contenant ladite fraction active
WO1993008264A1 (fr) * 1991-10-17 1993-04-29 Valio Bioproducts Ltd. Fraction du colostrum, sa preparation et son utilisation comme additif de bouillon de culture de cellules

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19619990A1 (de) * 1996-05-17 1997-11-20 Charlotte Adler Verfahren zur Herstellung von Kolostralmilchprodukten sowie deren Verwendung

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EP0692023A1 (fr) 1996-01-17
EE03162B1 (et) 1999-02-15
AU6209194A (en) 1994-10-24

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