Indian Patents. 277907:"A CELL CULTURE MEDIUM COMPRISING OF A CHITIN DERIVATIVE"

Title of Invention	"A CELL CULTURE MEDIUM COMPRISING OF A CHITIN DERIVATIVE"
Abstract	Present invention relates to a novel cell culture medium supplemented with an effective amount of chitin derivative. The present invention also provides a suitable alternative to Fetal Bovine Serum used in cell culture medium.

Full Text	Field of Invention: The present invention relates to a novel cell culture medium supplemented with an effective amount of chitin derivative. Prior Art: Cell culture media formulations are supplemented with a range of additives including not-well-defined components like fetal bovine serum (FBS), also known as fetal calf serum (PCS) or extracts from animal embryos, organs or glands. Serum provides a wide variety of macromolecular proteins, low molecular weight nutrients, carrier proteins for water insoluble components etc. for in vitro cell growth. However, considerable ethical concern has been raised about the harvest and collection of FBS. Fetal bovine serum is obtained from fetuses removed from healthy dams found pregnant at slaughter houses. The fetus is removed during evisceration and blood is extracted via cardiac puncture without anesthesia. Thus, there are increasing concerns about animal suffering inflicted during serum collection that add an ethical imperative to move away from the use of serum. Technical disadvantages of using FBS include the risk of transferable infectious organisms in mammalian plasma or serum products. Another serious drawback of serum products is the emergence of serious concerns for the transferable spongiform encephalopathy (TSE) such as 'mad cow disease' and the possibility of Prions or infectious proteins in plasma or serum derivatives. Also serum, in general is an ill-defined component in cell culture media. Batch to batch variation has also been reported. Also, PCS or FBS is the most costly component, about $ 456 / L (as per August 2000 price). Thus, in order to decrease the annual need for bovine fetuses in terms of the 3Rs ( Reduction, Refinement & Replacement) through any reduction in use or partial replacement of serum, as well as in terms of an improvement of cell and tissue culture methodology, other sources have been obtained. While FBS is the most commonly applied supplement in animal cell culture media, other sources are also routinely used, including newborn calf, horse and human organs or glands that have been used to prepare extracts for the supplementation of culture media include submaxillary gland ( Cohen,S., J.Bio.Chem.237:1555-1565( 1961), pituitary ( Peehl, D.M.,and Ham, R.G., Invitrol6:516-525 (1980), hypothalamus (Maciag, T., et al., Proc Natl. Acad. Sci. USA 76: 5674-5678(1979), Ocular retina and brain.( Maciag, T., et al., Science 211:1452-1454,(1981). Besides ethical concern batch to batch variations have been reported from even a single manufacturer. USP No. 5,426,045 and 5,443,984, relates to a method using fish whole serum to replace FBS or other animal serum. This fish serum provided the important advantage of a low probability of mammalian infectious agents. However, the fish whole serum was found to be toxic to many mammalian cells and ineffective for others. The other major drawback of USP No. 5,426,045 was the high lipid content of fish serum which was considered as 'potentially inhibiting' to mammalian cell growth. Yet another disadvantage of USP No. 5,426,045 is that even after removing the lipoprotein from the plasma, the material proved toxic to the mammalian cells. Focus on Alternatives [FOA], a group of British organizations working together to advance the replacement of animal experiments, has compiled a range of serum free media formulations (196 types). By definition Serum free media (SFM) should not contain whole serum as an ingredient, but it may not be entirely free from serum-derived products. FOA has not researched in detail the sources of other animal-derived ingredients with regard to animal welfare. Serum free media has several advantages over the serum containing media. However, the serum free media suffers from several disadvantages: • Investment and time is required to adopt a particular cell-line to serum free medium; • some cell-lines may require the addition of growth factor specific to that cell type to overcome a deficiency in the particular medium employed; • Low concentration of protein in the serum free medium makes the cell line vulnerable to shear forces and makes attachment to growth substratum difficult.; • Attachment dependent cell-lines require an extracellular matrix on the growth substratum. Thus, serum is needed to provide some components for this matrix; • Therefore, when using serum-free-medium the substratum ( plastic dishes) should be pre-coated with a fibronectin, laminin or another suitable alternative such as FNC coating mixture. Another prior art USP NO. 435069100 relates to the use of sodium butyrate and other salt of butyric acid to enhance the protein yield in animal cell-line in serum free media. However, the negative effect on growth rate can be very drastic even upon minor increase in concentrations. Yet, another art discloses a synergistic protein yield enhancing effects of Lithium salts or lipopolysaccharides in CHO cell-line, already comprising Sodium Butyrate (USP NO. 5378612). The art suffers from low yield (1.3 times). Another art USP No. 20050287666 describes cell culture media comprising transitional metals and other trace metals. The above art enlists a large number of transitional elements and no specific component is given. As each of these elements will have a different growth pattern and potential. Impurities even at a minuscule level may hinder the growth. Also quite high concentrations are recommended in the formulations. Thus, there still remains a need in the art for a suitable alternative to serum used in cell culture medium that permits the growth of the cells, is easily available, is economical and supports the ethical concern for the animals by completely eliminating the use of FBS. Object of the invention: The main object of the present invention is to provide an alternative to FBS used in cell culture medium. Another object of the invention is to provide a culture composition comprising a chitin derivative along with suitable cell culture medium. Summary of the invention; The present invention provides a novel cell culture medium supplemented with a Chitin derivative. Description of the accompanying drawings: Fig 1 represents the cell growth pattern in terms of cell count per unit area in three different media. Detailed description of the invention; Accordingly the present invention provides a novel cell culture medium supplemented with a chitin derivative. The said chitin derivative may be any derivative satisfying the following conditions: 1. has an amino group at position 2 of chitin monomer; 2. is a N-deacetylated derivative of chitin; 3. is water soluble. Surprisingly, a cell culture medium which is supplemented with an effective amount of a chitin derivative is found to show enhanced cell growth and cell viability. Therefore, the said chitin derivative may effectively be substituted for fetal bovine serum and other serum. Preferably the derivative is N-deacetylated carboxymethyl chitosan. Preferably, the amount of chitin derivative employed may be 0.5% to 10%. More preferably, the amount of derivative is 0.9% to 2%. In an embodiment, the chitin derivative may be formulated as a capsule or a tablet which may be readily used in the cell culture medium as supplement. The said formulation may contain an effective amount of the supplement/chitin derivative together with additives if any. The invention may be employed in any cell culture medium selected from the group comprising of Dulbecco's Modified Eagle Medium, Minimum Essential Medium Eagle, Basal Eagle Medium, Ham's F-10 medium, BGJb Medium and RPMI-1640. The invention is illustrated further by the following examples which are only meant to illustrate the invention and not act as limitations. The embodiments which may be apparent to a person skilled in the art are deemed to fall within the scope of the present invention. Example 1: Preparation of modified chitosan: Chitosan is treated with Sodium hydroxide in water-Isopropylalcohol medium at a temperature of 60°C. The causticized chitosan is then allowed to react with mono-chloric acetic acid. Precipitation is observed as a result of reaction. This precipitated mass is filtered and washed successively with water-alcohol mixture to make it free from salt and alkaline material followed by vacuum drying to get carboxymethyl chitosan (modified chitosan). Example 2: Preparation of stock of chitin derivative: For preparing 100ml of 10% (w/v) stock solution, lOg of said derivative is dissolved in small amount of solvent. Water is the most frequently used solvent. When the chitin derivative is dissolved properly, sufficient amount of solvent is added to make the solution 100ml followed by filtration through 0.22 ^ filter. The stock thus obtained does not require any further treatment and is stable for 2 weeks at room temperature. Example 3: 10% (w/v) modified chitosan is prepared using ultra pure water and filtering through 0.22 n filter. Dulbecco's Modified Eagle Medium [DMEM] Sigma, USA is supplemented with 1% modified (i.e. carboxymethyl) chitosan. (The composition of the DMEM is reproduced in Table 1) The solution is used to grow cells in a sterile 96 well flat bottom tissue culture plate. The cell lines selected for the purpose are BHK-21, HeLa, C6/36, Vero, L-132, BGM and like. Cells are kept in 5% CO2 tension at 37° C. The cell growth is monitored at 24 hrs and 48hrs for the control media, media containing FBS and media containing modified chitosan. The modified chitosan incorporated media shows better growth and longevity of the cells as compared to other two media preparations. The results are shown in Figure 1, wherein growth of cells in a medium supplemented with FBS is compared with medium supplemented with chitosan. The figure clearly shows that cell growth is higher in modified chitosan incorporated media. The viability of the cells was also higher in chitosan containing media as revealed by cell adhesion onto the surface of tissue culture flask, at different time intervals. This also clearly and unequivocally shows that the composition of the invention is synergistic as it actively promotes the growth of cells. Examples 4: 10% (w/v) unmodified chitosan is dissolved in acetic acid and filtered through 0.22 \i filter. From this stock solution 1% unmodified chitosan solution is incorporated in Dulbecco's Modified Eagle Medium [DMEM] Sigma, USA. The solution is used to grow cells in a sterile 96 well flat bottom tissue culture plate. The cell lines selected are BHK-21, HeLa C6/36, Vero, L-132, BGM and like. Cells are kept in 5% CO2 tension at 37° C. The cell growth is monitored at 24 and 48 hrs. For the control media, media containing FBS and media containing unmodified chitosan. The Chitosan incorporated media shows improved growth and longevity of the cells as compared to other two media preparations. TABLE 1; The most widely used modification is Dulbecco's Modified Eagle's Medium. DME is a modification of Minimum Essential Medium Eagle (MEM) that contains a higher concentration of vitamins and amino acids, as well as additional supplementary components g/1 0.2 0.0001 0.09767 0.4 6.4 0.109 0.084 0.0626 0.584 0.030 0.042 0.015 0.146 0.030 0.066 0.042 0.095 0.016 0.10379 0.094 0.004 0.004 0.0072 0.004 0.004 Components Calcium Chloride (anhydrous) Ferric Nitrate.9H2O Magnesium Sulfate (anhydrous) Potassium Chloride Sodium Chloride Sodium Phosphate Monobasic(anhy) L-Arginine. HCL L-Cystine.2HCl L-Glutamine Glycine L-Histidine.HCl.H2O L-Isoleucine L-Lysine.HCl L-Methionine L-Phenylalanine L-Serine L-Threonine L-Tryptophan L-Tyrosine.2Na.2H2O L-Valine Choline Chloride Folic acid myo-Inositol Nicotinamide Di antothenic Acid (Hemicalcium) Pyridoxal.HCl 0.004 Riboflavin 0.0004 Thiamine.HCl 0.004 D-Glucose 1.0 Phenol Red.Na 0.0159 Pyruvic Acid.Na 0.11 10% fetal bovine serum (FBS) is to be added to the contents mentioned above to make growth medium. Present invention replaces 10% FBS by 1% chitosan. Advantages of the present invention; Consequently, it can be observed that the cell culture media or the formulation of the present application is not a mere admixture having the aggregate properties of all the ingredients but act as a synergistic mixture resulting in enhanced cell growth and cell viability. The advantages of the present invention as under: (a) Provides a new growth supplement for the cell culture which is derived from an abundantly available natural resource, which is water soluble, biodegradable and can be used for a number of cell lines. (b) The cell culture media of the present invention shows better cell growth and cell viability as compared to medium containing fetal bovine serum. (c) The cell culture media of the present invention is simplified and better defined; have reduced degree of contamination; shows more consistent performance; have easier purification process and have no potential source of infection. (d)The new growth supplement i.e. chitosan (both modified as well as unmodified) solution is stable at room temperature for 2 weeks after filtration through 0.22 \i filter. Whereas fetal bovine serum (FBS) is required to be stored as -20°C all throughout. (e) The present invention addresses an important issue of the ethical concern by way of eliminating the need of animal killing. (f) Provides a very cost effective substitute for FBS containing media. The cost of modified chitosan is Rs 600/- per Kg. Since media requires 1% solution. The cost of Igm chitosan (for preparation of 1% solution) comes 60 paisa. On the other hand the cost of Fetal Bovine Serum (FBS) (Sigma Cat No F 2442) is Rs 24,000/- per 500 ml. 10% FBS is required to be supplemented in the media. Thus the cost of 10% FBS comes to Rs 480/-. Therefore the cell culture media of the present invention is economical and more viable. We Claim: 1. A medium supplemented with an effective amount of chitin derivative, wherein the said derivative is water soluble and has at least one amino group. 2. A medium as claimed in claim 1, wherein the derivative is N- deacetylated derivative of chitin. 3. A medium as claimed in claim 1, wherein the derivative has an amino group at position 2 of chitin monomer. 4. A medium as claimed in claims 2 or 3, wherein the derivative is chitosan. 5. A medium as claimed in claim 4, wherein the chitosan is modified chitosan. 6. A medium as claimed in claim 5, wherein the modified chitosan is N- deacetylated carboxymethyl chitosan. 7. A medium as claimed in any of claims 1-6, wherein the amount of chitin derivative is 0.5% to 10%. 8. A medium as claimed in claim 7, wherein the preferred amount of derivative is 0.9% to 2%. 9. A formulation comprising a chitin derivative together with additives. 10. A formulation as claimed in claim 9, wherein the formulation is in form of tablet or capsule. 11. A formulation as claimed in claim 1, wherein the medium is selected from the group comprising of Dulbecco's Modified Eagle Medium, Minimum Essential Medium Eagle, Basal Eagle Medium, Ham's F-10 medium, BGJb Medium and RPMI-1640. 12. A medium and a formulation such as herein described with * reference to the accompanying drawings and as illustrated in the foregoing examples.

Full Text

Field of Invention:
The present invention relates to a novel cell culture medium supplemented with an effective amount of chitin derivative.
Prior Art:
Cell culture media formulations are supplemented with a range of additives including not-well-defined components like fetal bovine serum (FBS), also known as fetal calf serum (PCS) or extracts from animal embryos, organs or glands. Serum provides a wide variety of macromolecular proteins, low molecular weight nutrients, carrier proteins for water insoluble components etc. for in vitro cell growth.
However, considerable ethical concern has been raised about the harvest and collection of FBS. Fetal bovine serum is obtained from fetuses removed from healthy dams found pregnant at slaughter houses. The fetus is removed during evisceration and blood is extracted via cardiac puncture without anesthesia. Thus, there are increasing concerns about animal suffering inflicted during serum collection that add an ethical imperative to move away from the use of serum.
Technical disadvantages of using FBS include the risk of transferable infectious organisms in mammalian plasma or serum products. Another serious drawback of serum products is the emergence of serious concerns for the transferable spongiform encephalopathy (TSE) such as 'mad cow disease' and the possibility of Prions or infectious proteins in plasma or serum derivatives. Also serum, in general is an ill-defined component in cell culture media. Batch to batch variation has also been reported. Also, PCS or FBS is the most costly component, about $ 456 / L (as per August 2000 price). Thus, in order to decrease the annual need for bovine fetuses in terms of the 3Rs ( Reduction, Refinement & Replacement) through any reduction in use or partial replacement of serum, as well as in terms of an improvement of cell and tissue culture methodology, other sources have been obtained.

While FBS is the most commonly applied supplement in animal cell culture media, other sources are also routinely used, including newborn calf, horse and human organs or glands that have been used to prepare extracts for the supplementation of culture media include submaxillary gland ( Cohen,S., J.Bio.Chem.237:1555-1565( 1961), pituitary ( Peehl, D.M.,and Ham, R.G., Invitrol6:516-525 (1980), hypothalamus (Maciag, T., et al., Proc Natl. Acad. Sci. USA 76: 5674-5678(1979), Ocular retina and brain.( Maciag, T., et al., Science 211:1452-1454,(1981). Besides ethical concern batch to batch variations have been reported from even a single manufacturer.
USP No. 5,426,045 and 5,443,984, relates to a method using fish whole serum to replace FBS or other animal serum. This fish serum provided the important advantage of a low probability of mammalian infectious agents. However, the fish whole serum was found to be toxic to many mammalian cells and ineffective for others. The other major drawback of USP No. 5,426,045 was the high lipid content of fish serum which was considered as 'potentially inhibiting' to mammalian cell growth. Yet another disadvantage of USP No. 5,426,045 is that even after removing the lipoprotein from the plasma, the material proved toxic to the mammalian cells.
Focus on Alternatives [FOA], a group of British organizations working together to advance the replacement of animal experiments, has compiled a range of serum free media formulations (196 types). By definition Serum free media (SFM) should not contain whole serum as an ingredient, but it may not be entirely free from serum-derived products. FOA has not researched in detail the sources of other animal-derived ingredients with regard to animal welfare.
Serum free media has several advantages over the serum containing media. However, the serum free media suffers from several disadvantages:

• Investment and time is required to adopt a particular cell-line to serum free medium;
• some cell-lines may require the addition of growth factor specific to that cell
type to overcome a deficiency in the particular medium employed;
• Low concentration of protein in the serum free medium makes the cell line
vulnerable to shear forces and makes attachment to growth substratum
difficult.;
• Attachment dependent cell-lines require an extracellular matrix on the
growth substratum. Thus, serum is needed to provide some components for
this matrix;
• Therefore, when using serum-free-medium the substratum ( plastic dishes)
should be pre-coated with a fibronectin, laminin or another suitable
alternative such as FNC coating mixture.
Another prior art USP NO. 435069100 relates to the use of sodium butyrate and other salt of butyric acid to enhance the protein yield in animal cell-line in serum free media. However, the negative effect on growth rate can be very drastic even upon minor increase in concentrations.
Yet, another art discloses a synergistic protein yield enhancing effects of Lithium salts or lipopolysaccharides in CHO cell-line, already comprising Sodium Butyrate (USP NO. 5378612). The art suffers from low yield (1.3 times).
Another art USP No. 20050287666 describes cell culture media comprising transitional metals and other trace metals. The above art enlists a large number of transitional elements and no specific component is given. As each of these elements will have a different growth pattern and potential. Impurities even at a minuscule level may hinder the growth. Also quite high concentrations are recommended in the formulations.

Thus, there still remains a need in the art for a suitable alternative to serum used in cell culture medium that permits the growth of the cells, is easily available, is economical and supports the ethical concern for the animals by completely eliminating the use of FBS.
Object of the invention:
The main object of the present invention is to provide an alternative to FBS
used in cell culture medium.
Another object of the invention is to provide a culture composition comprising a chitin derivative along with suitable cell culture medium.
Summary of the invention;
The present invention provides a novel cell culture medium supplemented with a Chitin derivative.
Description of the accompanying drawings:
Fig 1 represents the cell growth pattern in terms of cell count per unit area in three different media.
Detailed description of the invention;
Accordingly the present invention provides a novel cell culture medium supplemented with a chitin derivative. The said chitin derivative may be any derivative satisfying the following conditions:
1. has an amino group at position 2 of chitin monomer;
2. is a N-deacetylated derivative of chitin;
3. is water soluble.
Surprisingly, a cell culture medium which is supplemented with an effective amount of a chitin derivative is found to show enhanced cell growth and cell viability. Therefore, the said chitin derivative may effectively be substituted for fetal bovine serum and other serum. Preferably the derivative is N-deacetylated carboxymethyl chitosan.

Preferably, the amount of chitin derivative employed may be 0.5% to 10%.
More preferably, the amount of derivative is 0.9% to 2%.
In an embodiment, the chitin derivative may be formulated as a capsule or a
tablet which may be readily used in the cell culture medium as supplement.
The said formulation may contain an effective amount of the
supplement/chitin derivative together with additives if any.
The invention may be employed in any cell culture medium selected from the
group comprising of Dulbecco's Modified Eagle Medium, Minimum Essential
Medium Eagle, Basal Eagle Medium, Ham's F-10 medium, BGJb Medium
and RPMI-1640.
The invention is illustrated further by the following examples which are only meant to illustrate the invention and not act as limitations. The embodiments which may be apparent to a person skilled in the art are deemed to fall within the scope of the present invention.
Example 1: Preparation of modified chitosan: Chitosan is treated
with Sodium hydroxide in water-Isopropylalcohol medium at a temperature of 60°C. The causticized chitosan is then allowed to react with mono-chloric acetic acid. Precipitation is observed as a result of reaction. This precipitated mass is filtered and washed successively with water-alcohol mixture to make it free from salt and alkaline material followed by vacuum drying to get carboxymethyl chitosan (modified chitosan).
Example 2: Preparation of stock of chitin derivative: For preparing
100ml of 10% (w/v) stock solution, lOg of said derivative is dissolved in small amount of solvent. Water is the most frequently used solvent. When the chitin derivative is dissolved properly, sufficient amount of solvent is added to make the solution 100ml followed by filtration through 0.22 ^ filter. The stock thus obtained does not require any further treatment and is stable for 2 weeks at room temperature.

Example 3: 10% (w/v) modified chitosan is prepared using ultra pure
water and filtering through 0.22 n filter. Dulbecco's Modified Eagle Medium [DMEM] Sigma, USA is supplemented with 1% modified (i.e. carboxymethyl) chitosan. (The composition of the DMEM is reproduced in Table 1) The solution is used to grow cells in a sterile 96 well flat bottom tissue culture plate. The cell lines selected for the purpose are BHK-21, HeLa, C6/36, Vero, L-132, BGM and like. Cells are kept in 5% CO2 tension at 37° C. The cell growth is monitored at 24 hrs and 48hrs for the control media, media containing FBS and media containing modified chitosan. The modified chitosan incorporated media shows better growth and longevity of the cells as compared to other two media preparations.
The results are shown in Figure 1, wherein growth of cells in a medium supplemented with FBS is compared with medium supplemented with chitosan. The figure clearly shows that cell growth is higher in modified chitosan incorporated media. The viability of the cells was also higher in chitosan containing media as revealed by cell adhesion onto the surface of tissue culture flask, at different time intervals. This also clearly and unequivocally shows that the composition of the invention is synergistic as it actively promotes the growth of cells.
Examples 4: 10% (w/v) unmodified chitosan is dissolved in acetic acid
and filtered through 0.22 \i filter. From this stock solution 1% unmodified chitosan solution is incorporated in Dulbecco's Modified Eagle Medium [DMEM] Sigma, USA. The solution is used to grow cells in a sterile 96 well flat bottom tissue culture plate. The cell lines selected are BHK-21, HeLa C6/36, Vero, L-132, BGM and like. Cells are kept in 5% CO2 tension at 37° C. The cell growth is monitored at 24 and 48 hrs. For the control media, media containing FBS and media containing unmodified chitosan. The Chitosan incorporated media shows improved growth and longevity of the cells as compared to other two media preparations.

TABLE 1;
The most widely used modification is Dulbecco's Modified Eagle's Medium.
DME is a modification of Minimum Essential Medium Eagle (MEM) that
contains a higher concentration of vitamins and amino acids, as well as
additional supplementary components
g/1 0.2
0.0001
0.09767
0.4
6.4
0.109
0.084
0.0626
0.584
0.030
0.042
0.015
0.146
0.030
0.066
0.042
0.095
0.016
0.10379
0.094
0.004
0.004
0.0072
0.004
0.004
Components
Calcium Chloride (anhydrous)
Ferric Nitrate.9H2O
Magnesium Sulfate (anhydrous)
Potassium Chloride
Sodium Chloride
Sodium Phosphate Monobasic(anhy)
L-Arginine. HCL
L-Cystine.2HCl
L-Glutamine
Glycine
L-Histidine.HCl.H2O
L-Isoleucine
L-Lysine.HCl
L-Methionine
L-Phenylalanine
L-Serine
L-Threonine
L-Tryptophan
L-Tyrosine.2Na.2H2O
L-Valine
Choline Chloride
Folic acid
myo-Inositol
Nicotinamide
Di antothenic Acid (Hemicalcium)

Pyridoxal.HCl 0.004
Riboflavin 0.0004
Thiamine.HCl 0.004
D-Glucose 1.0
Phenol Red.Na 0.0159
Pyruvic Acid.Na 0.11
10% fetal bovine serum (FBS) is to be added to the contents mentioned above to make growth medium. Present invention replaces 10% FBS by 1% chitosan.
Advantages of the present invention;
Consequently, it can be observed that the cell culture media or the formulation of the present application is not a mere admixture having the aggregate properties of all the ingredients but act as a synergistic mixture resulting in enhanced cell growth and cell viability. The advantages of the present invention as under:
(a) Provides a new growth supplement for the cell culture which is
derived from an abundantly available natural resource, which is water
soluble, biodegradable and can be used for a number of cell lines.
(b) The cell culture media of the present invention shows better cell
growth and cell viability as compared to medium containing fetal
bovine serum.
(c) The cell culture media of the present invention is simplified and better
defined; have reduced degree of contamination; shows more consistent
performance; have easier purification process and have no potential
source of infection.
(d)The new growth supplement i.e. chitosan (both modified as well as unmodified) solution is stable at room temperature for 2 weeks after filtration through 0.22 \i filter. Whereas fetal bovine serum (FBS) is required to be stored as -20°C all throughout.
(e) The present invention addresses an important issue of the ethical
concern by way of eliminating the need of animal killing.
(f) Provides a very cost effective substitute for FBS containing media. The
cost of modified chitosan is Rs 600/- per Kg. Since media requires 1%
solution. The cost of Igm chitosan (for preparation of 1% solution)
comes 60 paisa. On the other hand the cost of Fetal Bovine Serum (FBS)
(Sigma Cat No F 2442) is Rs 24,000/- per 500 ml. 10% FBS is required to
be supplemented in the media. Thus the cost of 10% FBS comes to Rs
480/-. Therefore the cell culture media of the present invention is
economical and more viable.

We Claim:
1. A medium supplemented with an effective amount of chitin derivative,
wherein the said derivative is water soluble and has at least one amino
group.
2. A medium as claimed in claim 1, wherein the derivative is N-
deacetylated derivative of chitin.
3. A medium as claimed in claim 1, wherein the derivative has an amino
group at position 2 of chitin monomer.
4. A medium as claimed in claims 2 or 3, wherein the derivative is
chitosan.
5. A medium as claimed in claim 4, wherein the chitosan is modified
chitosan.
6. A medium as claimed in claim 5, wherein the modified chitosan is N-
deacetylated carboxymethyl chitosan.
7. A medium as claimed in any of claims 1-6, wherein the amount of
chitin derivative is 0.5% to 10%.
8. A medium as claimed in claim 7, wherein the preferred amount of
derivative is 0.9% to 2%.
9. A formulation comprising a chitin derivative together with additives.
10. A formulation as claimed in claim 9, wherein the formulation is in form
of tablet or capsule.
11. A formulation as claimed in claim 1, wherein the medium is selected
from the group comprising of Dulbecco's Modified Eagle Medium,
Minimum Essential Medium Eagle, Basal Eagle Medium, Ham's F-10
medium, BGJb Medium and RPMI-1640.
12. A medium and a formulation such as herein described with
*
reference to the accompanying drawings and as illustrated in the foregoing examples.

Documents:

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Patent Number

277907

Indian Patent Application Number

255/DEL/2007

PG Journal Number

51/2016

Publication Date

09-Dec-2016

Grant Date

05-Dec-2016

Date of Filing

07-Feb-2007

Name of Patentee

DIRECTOR GENERAL, DEFENCE RESEARCH & DEVELOPMENT ORGANISATION

Applicant Address

MINISTRY OF DEFENCE, GOVT OF INDIA, DIRECTORATE OF ER & IPR GROUP, ROOM NO. 348-B WING, DRDO BHAVAN, RAJAJI MARG, NEW DELHI 110 011

Inventors:

#	Inventor's Name	Inventor's Address
1	RAKESH BHARGAVA	DEFENCE R & D ESTABLISHMENT, JHANSI ROAD, GWALIOR-474 002
2	MD. SAFIKUMAR RAHMAN	AHMEDABAD TEXTILE INDUSTRY'S RESEARCH ASSOCIATION (ATIRA) AHMEDABAD-380 015
3	RAM SINGH CHAUHAN	DEFENCE R & D ESTABLISHMENT, JHANSI ROAD, GWALIOR-474 002
4	KRISHNAMURTHY SEKHAR	DEFENCE R & D ESTABLISHMENT, JHANSI ROAD, GWALIOR-474 002

PCT International Classification Number

C12N5/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA