Claims:1. A composition for in-vitro proliferation of human hair follicle stem cells, said composition comprising:
a. a first solution comprising:
i. inorganic salts selected from the group consisting of calcium chloride, ferric nitrate.9H2O, magnesium sulphate (anhydrous), potassium chloride, sodium chloride, sodium phosphate monobasic.H2O, and combinations thereof;
ii. amino acids selected from the group consisting of L-arginine.HCl, glycine, L-histidine.HCl.H2O, L-isoleucine, L-leucine, L-lysine.HCl, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine.2Na.2H2O, L-valine, L-cysteine.HCl, L-glutamine, L-methionine and combinations thereof;
iii. vitamins selected from the group consisting of choline chloride, folic acid, myo-inositol, niacinamide, D-pantothenic acid (hemicalcium), pyridoxine.HCl, riboflavin, thiamine.HCl, and combinations thereof; and
iv. D-glucose, sodium bicarbonate, and sodium pyruvate;
b. a second solution comprising:
i. inorganic salts selected from the group consisting of calcium chloride, cupric sulphate.5H2O, ferric sulphate.7H2O, magnesium chloride, potassium chloride, sodium chloride, sodium phosphate dibasic (anhydrous), zinc sulphate.7H2O, and combinations thereof;
ii. amino acids selected from the group consisting of L-alanine, L-arginine.HCl, L-asparagine.H2O, L-aspartic acid, L-cysteine.HCl.H2O, L-glutamic acid, glycine, L-histidine.3HCl.H2O, L-isoleucine, L-leucine, L-lysine.HCl, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine.2Na.2H2O, L-valine, L-glutamine, L-proline, and combinations thereof;
iii. vitamins selected from the group consisting of D-biotin, choline chloride, folic acid, myo-inositol, niacinamide, D-pantothenic acid (hemicalcium), pyridoxine.HCl, riboflavin, thiamine.HCl, vitamin B12, and combinations thereof; and
iv. D-glucose, hypoxanthine, linolenic acid, lipoic acid putrescine.HCl, pyruvic acid, Na pyruvate, sodium bicarbonate and thymidine; and
c. at least one growth factor selected from the group consisting of fibroblast growth factor, epidermal growth factor, nerve growth factor, insulin-like growth factor, platelet-derived growth factor, and combinations thereof,
wherein said first solution to second solution v/v ratio is 3:1, and said composition is serum free.
2. The composition as claimed in claim 1, wherein said at least one growth factor weight concentration in said composition is in the range of 20-40ng/ml of said composition.
3. The composition as claimed in claim 1, wherein said first solution comprises calcium chloride having weight concentration 0.2g/L, ferric nitrate.9H2O having weight concentration 0.0001g/L, magnesium sulphate (anhydrous) having weight concentration 0.09767g/L, potassium chloride having weight concentration 0.4g/L, sodium chloride having weight concentration 6.4g/L, and sodium phosphate monobasic having weight concentration 0.125g/L.
4. The composition as claimed in claim 1, wherein said first solution comprises L-arginine.HCl having weight concentration 0.084g/L, glycine having weight concentration 0.03g/L, L-histidine.HCl.H2O having weight concentration 0.042g/L, L-isoleucine having weight concentration 0.105g/L, L-leucine having weight concentration 0.105g/L, L-lysine.HCl having weight concentration 0.146g/L, L-phenylalanine having weight concentration 0.066g/L, L-serine having weight concentration 0.042g/L, L-threonine having weight concentration 0.095g/L, L-tryptophan having weight concentration 0.016g/L, L-tyrosine.2Na.2H2O having weight concentration 0.104g/L, and L-valine having weight concentration 0.094g/L, L-methionine having weight concentration 0.03g/L, L-serine having weight concentration 0.042g/L, L-cysteine.HCL having weight concentration 0.063g/L, and L-glutamine having weight concentration 0.584g/L.
5. The composition as claimed in claim 1, wherein said first solution comprises choline chloride having weight concentration 0.004g/L, folic acid having weight concentration 0.004g/L, myo-inositol having weight concentration 0.0072g/L, niacinamide having weight concentration 0.004g/L, D-pantothenic acid (hemicalcium) having weight concentration 0.004g/L, pyridoxine.HCl having weight concentration 0.004g/L, riboflavin having weight concentration 0.0004g/L, and thiamine.HCl having weight concentration 0.004g/L.
6. The composition as claimed in claim 1, wherein said first solution comprises D-glucose having weight concentration 4.5g/L, sodium pyruvate having weight concentration 0.11g/L, and sodium bicarbonate having weight concentration 13.7g/L.
7. The composition as claimed in claim 1, wherein said second solution comprises calcium chloride having weight concentration 0.0333g/L, cupric sulphate.5H2O having weight concentration 0.0000025g/L, ferric sulphate.7H2O having weight concentration 0.000834g/L, magnesium chloride having weight concentration 0.0576g/L, potassium chloride having weight concentration 0.224g/L, sodium phosphate dibasic.H2O having weight concentration 0.14204g/L, sodium chloride having weight concentration 7.599g/L, and zinc sulphate.7H2O having weight concentration 0.000863g/L.
8. The composition as claimed in claim 1, wherein said second solution comprises L-alanine having weight concentration 0.009g/L, L-arginine.HCl having weight concentration 0.211g/L, L-asparagine.H2O having weight concentration 0.01501g/L, L-aspartic acid having weight concentration 0.0133g/L, L-cysteine.HCl.H2O having weight concentration 0.035g/L, L-glutamic acid having weight concentration 0.0147g/L, glycine having weight concentration 0.00751g/L, L-histidine.3HCl.H2O having weight concentration 0.02096g/L, L-isoleucine having weight concentration 0.00394g/L, L-leucine having weight concentration 0.0131g/L, L-lysine.HCl having weight concentration 0.0365g/L, L-methionine having weight concentration 0.00448g/L, L-phenylalanine having weight concentration 0.00496g/L, L-serine having weight concentration 0.0105g/L, L-threonine, having weight concentration 0.0119g/L L-tryptophan having weight concentration 0.00204g/L, L-tyrosine.2Na.2H2O having weight concentration 0.00778g/L, L-glutamine having weight concentration 0.146g/L, L-proline having weight concentration 0.034g/L and L-valine having weight concentration 0.0117g/L.
9. The composition as claimed in claim 1, wherein said second solution comprises D-biotin having weight concentration 0.0000073g/L, choline chloride having weight concentration 0.01396g/L, folic acid having weight concentration 0.00132g/L, myo-inositol having weight concentration 0.018g/L, niacinamide having weight concentration 0.000037g/L, D-pantothenic acid (hemicalcium) having weight concentration 0.00048g/L, pyridoxine.HCl having weight concentration 0.000062g/L, riboflavin having weight concentration 0.000038g/L, thiamine.HCl having weight concentration 0.00034g/L, and vitamin B12 having weight concentration 0.00136g/L.
10. The composition as claimed in claim 1, wherein said second solution comprises D-glucose having weight concentration 1.802g/L, hypoxanthine having weight concentration 0.00478g/L, linolenic acid having weight concentration 0.000084g/L, putrescine.HCl having weight concentration 0.000161g/L, sodium pyruvate having weight concentration 0.11g/L, lipoic acid having weight concentration 0.00021g/L, sodium bicarbonate having weight concentration 1.176g/L and thymidine having weight concentration 0.00073g/L.
11. A composition for proliferation of human hair follicle stem cells, said composition comprising:
i. sodium chloride having weight concentration of 6.7g/L;
ii. potassium chloride having weight concentration of 0.356 g/L;
iii. disodium hydrogen phosphate having weight concentration of 0.035 g/L;
iv. sodium dihydrogen phosphate having weight concentration of 0.0937g/L
v. calcium chloride having weight concentration of 0.158 g/L;
vi. magnesium chloride having weight concentration of 0.0143 g/L;
vii. ferric sulphate. 7H2O having weight concentration of 0.0002 g/L;
viii. ferric nitrate. 9H2O having weight concentration of 0.0000075 g/L;
ix. magnesium sulphate having weight concentration of 0.0732 g/L;
x. cupric sulphate. 5H2O having weight concentration of 0.0000006g/L;
xi. zinc sulphate. 7H2O having weight concentration of 0.000215 g/L;
xii. L-alanine having weight concentration of 0.0025 g/L;
xiii. L-arginine having weight concentration of 0.115g/L;
xiv. L-asparagine.HCl having weight concentration of 0.003 g/L;
xv. L-aspartic acid having weight concentration of 0.003 g/L;
xvi. L-cysteine.HCl having weight concentration of 0.056 g/L;
xvii. L-glutamic acid having weight concentration of 0.0036 g/L;
xviii. L-glutamine having weight concentration of 0.474 g/L;
xix. glycine having weight concentration of 0.024 g/L;
xx. L-histidine.HCl. H2O having weight concentration of 0.036g/L;
xxi. L-isoleucine having weight concentration of 0.079 g/L;
xxii. L-leucine having weight concentration of 0.082 g/L;
xxiii. L-lysine.HCl having weight concentration of 0.118 g/L;
xxiv. L-methionine having weight concentration of 0.023 g/L;
xxv. L-phenylalanine having weight concentration of 0.050 g/L;
xxvi. L-proline having weight concentration of 0.008 g/L;
xxvii. L-serine having weight concentration of 0.034 g/L;
xxviii. L-threonine having weight concentration of 0.074 g/L;
xxix. L-tryptophan having weight concentration of 0.012 g/L;
xxx. L-tyrosine having weight concentration of 0.079 g/L;
xxxi. L-valine having weight concentration of 0.073 g/L;
xxxii. biotin having weight concentration of 0.00000182 g/ L;
xxxiii. D-Ca-panthothenate having weight concentration of 0.0031 g/L
xxxiv. choline chloride having weight concentration of 0.006 g/L;
xxxv. folic acid having weight concentration of 0.0033 g/L;
xxxvi. myo-inositol having weight concentration of 0.0099 g/L;
xxxvii. niacinamide having weight concentration of 0.00001 g/L;
xxxviii. pyridoxin.HCl having weight concentration of 0.003 g/L;
xxxix. riboflavin having weight concentration of 0.00031 g/L;
xl. thiamine.HCl having weight concentration of 0.0031 g/L;
xli. vitamin B12 having weight concentration of 0.00034 g/L;
xlii. hypoxanthine having weight concentration of 0.00119 g/L;
xliii. thymidine having weight concentration of 0.00018 g/L;
xliv. lipoic acid having weight concentration of 0.00005 g/L;
xlv. linoleic acid having weight concentration of 0.000021 g/L;
xlvi. glucose having weight concentration of 3.825 g/L;
xlvii. sodium pyruvate having weight concentration of 0.110 g/L;
xlviii. putrescine.2HCl having weight concentration of 0.000041 g/L;
xlix. basic fibroblast growth factor having weight concentration of 0.00002 g/L, and
l. sodium bicarbonate having weight concentration of 10.569 g/L
12. A method of promoting in-vitro proliferation of human hair follicle stem cells, said method comprising:
a. obtaining a composition as claimed in any of the claims 1-11; and
b. obtaining human hair follicle stem cells; and
c. incubating said human hair follicle stem cells in said composition,
wherein said method promotes in-vitro proliferation of human hair follicle stem cells in a serum free environment.
13. A method for in-vitro production and sustenance of colony forming units from human hair follicle stem cells, said method comprising:
a. obtaining a composition as claimed in any of the claims 1-11;
b. obtaining human hair follicle stem cells; and
c. incubating said human hair follicle stem cells in said composition,
wherein said method promotes production and sustenance of colony forming units from human hair follicle stems cells in a serum free environment.
, Description:As Attached