SERUM-FREE CULTURE MEDIUM FOR LIMBAL STEM CELLS AND CULTURE METHOD THEREOF

Abstract

A serum-free culture medium for limbal stem cells and a culture method thereof, wherein the serum-free culture medium includes a basic medium and supplements; wherein the supplements include: human recombinant EGF, insulin, 3,3,5-triiodo-L-thyronine, hydrocortisone, forskolin, manganese sulfate monohydrate, sodium selenite, sodium metasilicate, ammonium metavanadate, nickel chloride hexahydrate, stannous chloride dihydrate, ethanolamine, O-phosphorylethanolamine, ammonium molybdate tetrahydrate, 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid, vitamin C, bovine serum albumin, lipid concentrate and serum substitute. The serum-free culture medium according to the present invention is free of fetal bovine serum and any animal-derived ingredient, can provide necessary adequate nutrition and good environment for cell growth and proliferation, effectively replaces the role of serum, realizes favorable cell growth, improves the cell purity and stability, provides quick and stable cell sources for researches on the mechanism of limbal stem cell specificity and transplantation therapy, and has broad clinical application prospects.

Claims

1. A serum-free culture medium for limbal stem cells, comprising a basic medium and supplements; wherein the supplements comprise following ingredients by concentration: 10-20 ng/mL of an human recombinant EGF, 5-10 g/mL of insulin, 110.sup.9-510.sup.9 M of 3,3,5-triiodo-L-thyronine, 0.2-1 g/mL of hydrocortisone, 0.510.sup.5-210.sup.5 M of a forskolin, 0.510.sup.9-210.sup.9 M of manganese sulfate monohydrate, 510.sup.7-1010.sup.7 M of sodium selenite, 0.110.sup.3-110.sup.3 M of sodium metasilicate, 310.sup.6-810.sup.6 M of ammonium metavanadate, 310.sup.10-810.sup.10 M of nickel chloride hexahydrate, 310.sup.10-810.sup.10 M of stannous chloride dihydrate, 310.sup.7-810.sup.7 M of ethanolamine, 310.sup.6-810.sup.6 M of O-phosphorylethanolamine, 110.sup.9-610.sup.9 M of ammonium molybdate tetrahydrate, 2-8 g/L of 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid, 20-50 g/mL of vitamin C, 1%-3% of a bovine serum albumin, 0.5%-2% of a lipid concentrate, and 10%-15% of a serum substitute.

2. The serum-free culture medium for limbal stem cells according to claim 1, wherein concentrations of the ingredients in the supplements of the serum-free culture medium are as follows: 10 ng/mL of the human recombinant EGF, 5 g/mL of insulin, 2.510.sup.9 M of 3,3,5-triiodo-L-thyronine, 0.4 g/mL of hydrocortisone, 110.sup.5 M of the forskolin, 110.sup.9 M of manganese sulfate monohydrate, 610.sup.7 M of sodium selenite, 0.510.sup.3 M of sodium metasilicate, 510.sup.6 M of ammonium metavanadate, 510.sup.10 M of nickel chloride hexahydrate, 510.sup.10 M of stannous chloride dihydrate, 510.sup.7 M of ethanolamine, 510.sup.6 M of O-phosphorylethanolamine, 310.sup.9 M of ammonium molybdate tetrahydrate, 5.4 g/L of 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid, 30 g/mL of vitamin C, 2% of the bovine serum albumin, 1% of the lipid concentrate, and 10% of the serum substitute.

3. The serum-free culture medium for limbal stem cells according to claim 1, wherein the serum-free culture medium further comprises an antibiotic.

4. The serum-free culture medium for limbal stem cells according to claim 3, wherein the antibiotic is a penicillin-streptomycin solution.

5. The serum-free culture medium for limbal stem cells according to claim 1, wherein the basic medium is a DMEM/Ham's F12 medium.

6. The serum-free culture medium for limbal stem cells according to claim 5, wherein a volume ratio of the DMEM to Ham's F12 is 1:1.

7. An isolation and culture method of limbal stem cells, comprising the steps of: washing a corneal limbus tissue, enzymolyzing the corneal limbus tissue, and culturing a product after the enzymolyzing in the serum-free culture medium, wherein the serum-free culture medium comprising a basic medium and supplements, the supplements comprise following ingredients by concentration: 10-20 ng/mL of an human recombinant EGF, 5-10 g/mL of insulin, 110.sup.9-510.sup.9 M of 3,3,5-triiodo-L-thyronine, 0.2-1 g/mL of hydrocortisone, 0.510.sup.5-210.sup.5 M of a forskolin, 0.510.sup.9-210.sup.9 M of manganese sulfate monohydrate, 510.sup.7-1010.sup.7 M of sodium selenite, 0.110.sup.3-110.sup.3 M of sodium metasilicate, 310.sup.6-810.sup.6 M of ammonium metavanadate, 310.sup.10-810.sup.10 M of nickel chloride hexahydrate, 310.sup.10-810.sup.10 M of stannous chloride dihydrate, 310.sup.7-810.sup.7 M of ethanolamine, 310.sup.6-810.sup.6 M of O-phosphorylethanolamine, 110.sup.9-610.sup.9 M of ammonium molybdate tetrahydrate, 2-8 g/L of 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid, 20-50 g/mL of vitamin C, 1%-3% of a bovine serum albumin, 0.5%-2% of a lipid concentrate, and 10%-15% of a serum substitute.

8. The isolation and culture method according to claim 7, wherein the enzymolyzing is one-time enzymolyzing, and the enzymolyzing is carried out directly using a type IV collagenase.

9. The isolation and culture method according to claim 8, wherein a concentration of the type IV collagenase is 0.1%-0.5%; and the enzymolyzing lasts for 30-60 minutes.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIG. 1 shows a serum-free culture of human limbal stem cells (morphological features under a microscope).

[0037] FIG. 2A to FIG. 2C show an immunochemical staining of limbal stem cells specifically labeled with PAX6 antibody; wherein FIG. 2A is DAPI; FIG. 2B is PAX6; and FIG. 2C is after fusion.

[0038] FIG. 3A to FIG. 3C show an immunochemical staining of limbal stem cells specifically labeled with p63 antibody; wherein FIG. 3A is DAPI; FIG. 3B is p63; and FIG. 3C is after fusion.

[0039] FIG. 4A to FIG. 4C show immunochemical staining of limbal stem cells specifically labeled with Ki67 antibody; wherein FIG. 4A is DAPI; FIG. 4B is Ki67; and FIG. 4C is after fusion.

[0040] FIG. 5A to FIG. 5C show immunochemical staining of limbal stem cells specifically labeled with K12 antibody; wherein FIG. 5A is DAPI; FIG. 5B is K12; and FIG. 5C is after fusion.

[0041] FIG. 6 shows morphological features of cells cultured in the serum-free culture medium according to the present invention for 4 days.

[0042] FIG. 7 shows morphological features of cells cultured in the serum-free culture medium according to the present invention for 8 days.

[0043] FIG. 8 shows morphological features of cells cultured in the serum-free culture medium according to the present invention for 20 days.

[0044] FIG. 9 shows morphological features of P1 generation LSCs subcultured in the serum-free culture medium according to the present invention.

[0045] FIG. 10 shows an FBS-containing culture medium for human limbal stem cells (morphological features under a microscope).

DESCRIPTION OF THE EMBODIMENTS

[0046] The present invention is further described below in combination with specific examples, but the examples do not limit the present invention in any way. Without departing from the spirit and essence of the present invention, all simple modifications or replacements of the methods, steps or conditions of the present invention fall within the scope of the present invention. Unless otherwise specified, the technical means used in the examples are the conventional means known to those skilled in the art.

[0047] Unless otherwise stated, all reagents and materials used in the following examples are available on the market.

[0048] Collagen I: dissolving 1 mL of a collagen I in 9 mL of DMEM/Ham's F12 medium.

[0049] Type IV collagenase: 5 mg/mL of a type IV collagenase prepared from DMEM/Ham's F12 medium.

[0050] Stock solution of human recombinant EGF: 10 g/mL EGF stock solution prepared from PBS.

[0051] Stock solution of insulin: prepared from 0.005 mol/L HCl, with a concentration of 5 mg/mL.

[0052] Stock solution of 3,3,5-triiodo-L-thyronine: dissolving 13.6 mg of 3,3,5-iodothyronine in 15 mL of 0.02 mol/L NaOH solution, adding 85 mL of PBS; and diluting 0.1 mL of the prepared solution with PBS to 20 mL as the stock solution with a concentration of 110.sup.6 M.

[0053] Stock solution of hydrocortisone: dissolving 5 mg of hydrocortisone in 1 mL of anhydrous ethanol, and adding 400 L of the resulting solution to 10 mL of PBS to obtain the stock solution.

[0054] Stock solution of manganese sulfate monohydrate: dissolving 1.69 mg of manganese sulfate monohydrate in 10 mL of PBS to obtain a 10.sup.6-fold stock solution at a concentration of 110.sup.3 M.

[0055] Stock solution of sodium metasilicate: dissolving 0.61 g of sodium metasilicate in 10 mL of PBS to obtain a 1000-fold stock solution at a concentration of 0.5 M.

[0056] Stock solution of sodium selenite: dissolving 1.038 g of sodium selenite in 10 mL of PBS to obtain a 10.sup.6-fold stock solution at a concentration of 0.6 M.

[0057] Stock solution of ammonium metavanadate: dissolving 5.85 g of ammonium metavanadate in 10 mL of PBS to obtain a 10.sup.6-fold stock solution at a concentration of 5 M.

[0058] Stock solution of nickel chloride hexahydrate: dissolving 1.19 mg of nickel chloride hexahydrate in 10 mL of PBS to obtain a 10.sup.6-fold stock solution at a concentration of 510.sup.4 M.

[0059] Stock solution of stannous chloride dihydrate: dissolving 1.13 mg of stannous chloride dihydrate in 10 mL of PBS to obtain a 10.sup.6-fold stock solution at a concentration of 510.sup.4 M.

[0060] Stock solution of ethanolamine: dissolving 305 mg of ethanolamine in 10 mL of PBS to obtain a 10.sup.6-fold stock solution at a concentration of 0.5 M.

[0061] Stock solution of O-phosphoiylethanolamine: dissolving 7 mg of O-phosphorylethanolamine in 10 mL of PBS to obtain a 1000-fold stock solution at a concentration of 510.sup.3 M.

[0062] Stock solution of ammonium molybdate tetrahydrate: dissolving 37.08 mg of ammonium molybdate tetrahydrate in 10 mL of PBS to obtain a 10.sup.6-fold stock solution at a concentration of 310.sup.3 M.

[0063] Stock solution of vitamin C: dissolving 300 mg of vitamin C in 10 mL of PBS to obtain a 1000-fold stock solution at a concentration of 30 mg/mL.

EXAMPLE 1

[0064] 1. A Serum-Free Culture Medium for Limbal Stem Cells

[0065] A serum-free culture medium for limbal stem cells consists of following ingredients:

[0066] 216 mL of a Ham's F12, 216 mL of a DMEM, 5 mL of 100penicillin-streptomycin solution, 1 mL of a stock solution of a human recombinant EGF, 500 L of a stock solution of insulin, 500 L of a stock solution of vitamin C, 500 L of a stock solution of 3,3,5-triiodo-L-thyronine, 500 L of a stock solution of hydrocortisone, 1 L of a stock solution of a forskolin, 0.5 L of manganese sulfate monohydrate, 0.5 L of sodium selenite, 500 L of sodium metasilicate, 0.5 L of ammonium metavanadate, 0.5 L of nickel chloride hexahydrate, 0.5 L of stannous chloride dihydrate, 0.5 L of ethanolamine, 500 L of O-phosphorylethanolamine, 0.5 L of ammonium molybdate tetrahydrate, 2.7 g of 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid, 10 g of a bovine serum albumin, 5 mL of a lipid concentrate, and 50 mL of a serum substitute.

[0067] Concentrations of supplements are respectively: 10 ng/mL of the human recombinant EGF, 5 g/mL of insulin, 2.510.sup.9 M of 3,3,5-triiodo-L-thyronine, 0.4 g/mL of hydrocortisone, 110.sup.5 M of the forskolin, 110.sup.9 M of manganese sulfate monohydrate, 610.sup.7 M of sodium selenite, 0.510.sup.3 M of sodium metasilicate, 510.sup.6 M of ammonium metavanadate, 510.sup.10 M of nickel chloride hexahydrate, 510.sup.10 M of stannous chloride dihydrate, 510.sup.7 M of ethanolamine, 510.sup.6 M of O-phosphorylethanolamine, 310.sup.9 M of ammonium molybdate tetrahydrate, 5.4 g/L of 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid, 30 g/mL of vitamin C, 2% of the bovine serum albumin, 1% of the lipid concentrate, and 10% of the serum substitute.

[0068] 2. An Isolation and Culture Method of Limbal Stem Cells

[0069] (1) Under an operating microscope, a human corneal limbus tissue was cut with a tissue forcep and corneal scissors, and washed with a PBS containing a penicillin-streptomycin solution (1) in a sterile environment twice (5 minutes each time); and then cut into pieces with the scissors.

[0070] (2) According to a volume of a tissue block, 5 mL of 0.5% type IV collagenase solution was added to every 1 cm.sup.3 of the tissue block, digested while gently oscillating at 37 C. for 45 minutes, neutralized with 10-fold volume of the serum-free culture medium, and centrifuged at 1000 rpm for 5 minutes. Then the supernatant was discarded.

[0071] (3) After a 12-well plate was coated with 10% matrigel, centrifuged cells were resuspended in the serum-free culture medium, respectively cultivated in the wells of the 12-well plate, and cultured in an incubator at 37 C. under an atmosphere containing 5% CO.sub.2. The solution was replaced every other day, and the growth status of the cells was observed under a microscope.

[0072] 3. Analysis on the Experimental Results:

[0073] (1) FIG. 1 shows serum-free culture of human limbal stem cells. The results showed that the cultivated cells have the morphological structure of limbal stem cells, proving that the limbal stern cells can really be cultured in the serum-free culture medium.

[0074] (2) FIGS. 2A, 2B and 2C show immunochemical staining of limbal stem cells specifically labeled with PAX6 antibody; wherein blue is DAPI, and green is PAX6. The results show that the cultured cells are PAX6 positive, have the characteristics of limbal stem cells, and have a PAX6 antibody positive rate of 96%-100%.

[0075] (3) FIGS. 3A, 3B and 3C show immunochemical staining of stem cells specifically labeled with p63 antibody; wherein blue is DAPI, and green is p63. The results show that the cultured cells are p63 positive, have the characteristics of stem cells, and have a p63 antibody positive rate of 96%-100%.

[0076] (4) FIGS. 4A, 4B and 4C show immunochemical staining of limbal stem cells specifically labeled with Ki67 antibody; wherein blue is DAPI, and green is Ki67. The results show that the cultured cells are Ki67 positive, have the characteristics of limbal stem cells, and have a Ki67 antibody positive rate of 96%-100%.

[0077] (5) FIGS. 5A, 5B and 5C show immunochemical staining of limbal stem cells specifically labeled with K12 antibody; wherein blue is DAPI, and green is K12. The results show that the cultured cells are K12 positive, have the characteristics of limbal stem cells, and have a K12 antibody positive rate of 96%-100%.

[0078] (6) FIGS. 6-9 show the morphological features of cells cultured in the serum-free culture medium according to the present invention for 4-20 days and the morphological features of subcultured cells. The results show that the morphological features of cells comply with the characteristics of limbal stem cells.

[0079] As can be concluded from the above results, the serum-free culture medium according to the present invention can replace a serum-containing medium very well, and realize limbal stem cells culture and favorable cells growth. In the culture process, the limbal stem cells have fast proliferation, high purity, high activity and good stability; homogeneous limbal stem cells with an efficient in vitro amplification function have been obtained; and the cells quality has been improved, thereby providing a more quick and stable cell source for researches on the mechanism of limbal stem cell specificity and transplantation therapy.

EXAMPLE 2

Culture Medium Formula

[0080] The experimental method is identical to that in Example 1 only except that the concentrations of the supplements in the serum-free culture medium for limbal stem cells in the example are as follows:

[0081] 10 ng/mL of the human recombinant EGF, 10 g/mL of insulin, 510.sup.9 M of 3-iodothyronine, 0.2 g/mL of hydrocortisone, 210.sup.5 M of the forskolin, 0.510.sup.9 M of manganese sulfate monohydrate, 510.sup.7 M of sodium selenite, 0.110.sup.3 M of sodium metasilicate, 810.sup.6 M of ammonium metavanadate, 310.sup.10 M of nickel chloride hexahydrate, 810.sup.10 M of stannous chloride dihydrate, 310.sup.7 M of ethanolamine, 810.sup.6 M of O-phosphorylethanolamine, 110.sup.9 M of ammonium molybdate tetrahydrate, 8 g/L of 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid, 20 g/mL of vitamin C, 3% of the bovine serum albumin, 0.5% of lipid concentrate, and 15% of the serum substitute.

EXAMPLE 3

Culture Medium Formula

[0082] The experimental method is identical to that in Example 1 only except that the concentrations of the supplements in the serum-free culture medium for limbal stem cells in the example are as follows:

[0083] 20 ng/mL of the human recombinant EGF, 5 g/mL of insulin, 110.sup.9 M of 3-iodothyronine, 1 g/mL of hydrocortisone, 0.510.sup.5 M of the forskolin, 210.sup.9 M of manganese sulfate monohydrate, 1010.sup.7 M of sodium selenite, 110.sup.3 M of sodium metasilicate, 310.sup.6 M of ammonium metavanadate, 810.sup.10 M of nickel chloride hexahydrate, 310.sup.10 M of stannous chloride dihydrate, 810.sup.7 M of ethanolamine, 310.sup.6 M of O-phosphorylethanolamine, 610.sup.9 M of ammonium molybdate tetrahydrate, 2 g/L of 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid, 50 g/mL of vitamin C, 1% of the bovine serum albumin, 2% of the lipid concentrate, and 5% of the serum substitute.

COMPARISON EXAMPLE 1

Positive Control (Culture Medium Containing Fetal Bovine Serum)

[0084] The experimental method is identical to that in Example 1 only except that the medium for limbal stem cells in the example contains serum, and specifically includes the following ingredients: 216 mL of the Ham's F12, 216 mL of the DMEM, 5 mL of 100penicillin-streptomycin solution and 63 mL of the fetal bovine serum.

[0085] FIG. 10 shows a serum-containing culture of human limbal stem cells. It is found through researches that the limbal stem cells obtained from the serum-free culture medium in Example 1 are not significantly different from those obtained from the serum-containing medium; and the limbal stem cells in the serum-free culture medium have almost equivalent growth rate to those in the serum-containing medium, and have regular and uniform morphological features, suggesting that the limbal stem cells cultured in the serum-free culture medium according to the present invention also have very high purity. The results show that the serum-free culture medium can completely replace the serum-containing medium in culturing limal stem cells.

COMPARISON EXAMPLE 2

Negative Control (Only Containing a Basic Medium and Antibiotics)

[0086] The experimental method is identical to that in Example 1 only except that the medium for limbal stem cells in the example contains 216 mL of the Ham's F12, 216 mL of the DMEM, and 5 mL of 100penicillin-streptomycin solution.

[0087] It is found through researches that compared with the serum-free culture medium in Example 1, the limbal stem cells completely fail to grow adherently. The results show that this medium is not suitable for culturing limbal stem cells.

COMPARISON EXAMPLE 3

Medium Containing a Basic Medium, Antibiotics and a Serum Substitute

[0088] The experimental method is identical to that in Example 1 only except that the medium for limbal stem cells in the example contains 216 mL of the Ham's F12, 216 mL of the DMEM, 5 mL of 100penicillin-streptomycin solution and 10% of the serum substitute.

[0089] The results show that compared with the serum-free culture medium in Example 1, there is poor limbal stem cells growth status. Therefore, the medium is not suitable for culturing limbal stem cells.

COMPARISON EXAMPLE 4

[0090] The conditions are identical to those in Example 1, except that a concentration of the serum substitute is 1%.

[0091] The results show that when the concentration of the serum substitute is 1%, and the concentration of the serum substitute is very low, which can lead morphological features of the limbal stem cells to changing.

COMPARISON EXAMPLE 5

[0092] The conditions are identical to those in Example 1, except that a concentration of forskolin is 0.110.sup.5 M.

[0093] The results show that when the serum concentration is reduced from 110.sup.5 M to 0.110.sup.5 M, the limbal stem cells become larger, their cell nuclei become smaller, and their cell adherence capacity becomes worse, etc.

COMPARISON EXAMPLE 6

[0094] The conditions are identical to those in Example 1, except that a concentration of the lipid concentrate is 0.1%.

[0095] The results show that the concentration of the lipid concentrate is 0.1%, which can lead the limbal stem cells to growing slowly, the epidermal cells doing not have obvious morphological features, and there are loose intercellular connections of limbal stem cells and many dead cells in the serum-free culture medium.

[0096] Furthermore, it is found that the human recombinant EGF, insulin, 3-iodo-L-thyronine, hydrocortisone, the forskolin, manganese sulfate monohydrate, sodium selenite, sodium metasilicate, ammonium metavanadate, nickel chloride hexahydrate, stannous chloride dihydrate, ethanolamine, O-phosphorylethanolamine, ammonium molybdate tetrahydrate, 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid, vitamin C, the bovine serum albumin, the lipid concentrate and the serum substitute all play an important role in culturing limbal stem cells, and none thereof is dispensable.