CN112608898B - Corneal endothelial cell induction method - Google Patents
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Abstract
The invention relates to the technical field of cell culture. The invention provides a corneal endothelial cell induction method, which comprises the following steps of (1) culturing induced pluripotent stem cells in an mTeSR1 culture medium, a dual Smad inhibitor culture medium and a first corneal endothelial cell culture medium in sequence to obtain differentiated induced pluripotent stem cells; (2) Digesting the differentiated induced pluripotent stem cells, and blowing off the cells by using a second corneal endothelial cell culture medium to obtain a cell suspension; (3) And culturing the cell suspension with a mixed culture medium to obtain the corneal endothelial cells. The induction method of the invention can obtain a large amount of high-purity corneal endothelial cells in a short time, and the obtained corneal endothelial cells can be passaged in vitro for at least 4 times, thereby effectively solving the problems of limited number of human corneal donors and limited in vitro amplification capacity of primary corneal endothelial cells.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to a corneal endothelial cell induction method.
Background
The cornea consists of three cellular layers, an epithelial layer, a stromal layer and an endothelial layer, and the corneal endothelium is a 4 micron thick monolayer membrane located inside the cornea and responsible for maintaining corneal transparency by dehydrating the stroma. Human corneal endothelial cells cannot be regenerated in vivo, and the density of corneal endothelial cells gradually decreases with age, from about 4000/mm in a newborn 2 Reduced to about 2300/mm for the elderly 2 . When the corneal endothelial cell density reaches a critically low level, the stromal layer may edema, causing pain and producing corneal blindness.
Full transplantation of all corneal layers and selective replacement of only the damaged endothelial layer, with new and improved approaches such as Descemet's membrane endothelial keratoplasty and Descemet's membrane endothelial transfer being the primary surgical treatment option for corneal endothelial dysfunction. In recent years, the culture of primary human corneal endothelial cells has made new progress; however, the number of human corneal donors is quite limited, and primary sources of human corneal endothelial cells have limited ability to expand in vitro.
Disclosure of Invention
The invention aims to provide a corneal endothelial cell induction method, which solves the problems of limited number of human corneal donors and limited in vitro amplification capacity of primary-source human corneal endothelial cells.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a corneal endothelial cell induction method, which comprises the following steps:
(1) Culturing the induced pluripotent stem cells in an mTeSR1 culture medium, a dual Smad inhibitor culture medium and a first corneal endothelial cell culture medium in sequence to obtain differentiated induced pluripotent stem cells;
(2) Digesting the differentiated induced pluripotent stem cells, and blowing off the cells by using a second corneal endothelial cell culture medium to obtain a cell suspension;
(3) And culturing the cell suspension with a mixed culture medium to obtain the corneal endothelial cells.
Preferably, the induced pluripotent stem cells are cultured in the mTeSR1 medium for 3 to 5 days.
Preferably, the induced pluripotent stem cells are cultured in a dual Smad inhibitor medium for 2 to 4 days, and the dual Smad inhibitor medium is prepared by the following steps: preparing a basic culture medium according to the volume ratio of 68.9-84.1 parts of DMEM/F12 culture medium, 15-30 parts of KSR and 0.9-1.1 parts of NEAA, and adding human recombinant Noggin, SB431542, glutamine, beta-mercaptoethanol and bFGF into the basic culture medium to obtain the basic culture medium containing 300-500 ng/mL of human recombinant Noggin, 9-11 mu M SB431542, 0.9-1.1 mM of glutamine, 0.09-0.11 mM of beta-mercaptoethanol and 5-20 ng/mL of bFGF, namely the dual Smad inhibitor culture medium.
Preferably, the induced pluripotent stem cells are cultured in a first corneal endothelial cell culture medium for 1 to 3 days, and the first corneal endothelial cell culture medium is prepared by a method comprising: preparing a basal culture medium according to the volume ratio of 68.9-84.1 parts of DMEM/F12 culture medium, 15-30 parts of KSR and 0.9-1.1 parts of NEAA, adding a B27 supplement, PDGF-BB, DKK-2, L-glutamine, beta-mercaptoethanol and bFGF into the basal culture medium to obtain the basal culture medium containing 0.09-0.11 XB 27 supplement, 9-11 ng/mLPDGF-BB, 9-11 ng/mL DKK-2, 0.8-1.2 mM L-glutamine, 0.09-0.11 mM beta-mercaptoethanol and 5-20 ng/mLbFGF, namely the first corneal endothelial cell culture medium.
Preferably, the digesting process is carried out by using Accutase, the digesting temperature is 35-39 ℃, and the digesting time is 3-7 min.
Preferably, the digested liquid is removed before the second corneal endothelial cell culture medium is added.
Preferably, the mixed culture medium is prepared by mixing a second corneal endothelial cell culture medium and a corneal endothelial cell conditioned medium according to a volume ratio of 1: 0.8-1.2, and the culture time in the mixed culture medium is 6-8 days.
Preferably, the volume ratio of the cell suspension to the mixed culture medium in the step (3) is 1:1.8 to 2.2.
Preferably, the second corneal endothelial cell culture medium is prepared by the following method: preparing a basic culture medium according to the volume ratio of DMEM/F1245-47 parts, M19945-47 parts, FBS 4.5-5.5 parts, NEAA 0.8-1.2 parts, glutaMAX 0.8-1.2 parts and ITS 0.8-1.2 parts, and adding bFGF and penillin-streptomycin into the basic culture medium to obtain the basic culture medium containing 5-20 ng/mLbFGF and 95-105 units/ml penillin-streptomycin, namely the second corneal endothelial cell culture medium.
Preferably, the preparation method of the corneal endothelial cell conditioned medium comprises the following steps: digesting the elastic layer behind the cornea to obtain a corneal endothelial cell cluster, continuously digesting to obtain a single cell, culturing by using a second corneal endothelial culture medium until the cell is fused, subculturing to P3 generation, and collecting culture supernatant of the P1-P3 generation, namely the corneal endothelial cell conditioned medium.
The invention provides a corneal endothelial cell induction method, which comprises the following steps of (1) culturing induced pluripotent stem cells in an mTeSR1 culture medium, a dual Smad inhibitor culture medium and a first corneal endothelial cell culture medium in sequence to obtain differentiated induced pluripotent stem cells; (2) Digesting the differentiated induced pluripotent stem cells, and blowing off the cells by using a second corneal endothelial cell culture medium to obtain a cell suspension; (3) And culturing the cell suspension with a mixed culture medium to obtain the corneal endothelial cells. The induction method of the invention can obtain a large amount of high-purity corneal endothelial cells in a short time, and the obtained corneal endothelial cells can be passaged in vitro for at least 4 times, thereby effectively solving the problems of limited number of human corneal donors and limited in vitro amplification capacity of primary corneal endothelial cells.
Drawings
FIG. 1 shows cells grown in the medium mTeSR1 for 4 days in example 2;
FIG. 2 is a photograph of cells grown in first corneal endothelial cell culture medium for 2 days in example 2;
FIG. 3 shows cells grown in mixed media for 7 days in example 2;
FIG. 4 shows P2 generation cells passaged by corneal endothelial cells induced in example 2;
FIG. 5 shows P3 generation cells passaged by corneal endothelial cells induced in example 2;
FIG. 6 shows cells grown in mTeSR1 medium for 4 days in example 5;
FIG. 7 shows P2 generation cells passaged by corneal endothelial cells induced in example 5;
FIG. 8 shows cells grown in the medium mTeSR1 for 4 days in example 6;
FIG. 9 shows P3 generation cells passaged by corneal endothelial cells induced in example 6;
FIG. 10 shows the cells passaged in example 7;
FIG. 11 shows immunofluorescence staining results of a marker Na-K-ATPase for corneal endothelial cells obtained in example 8;
FIG. 12 shows ZO-1 immunofluorescent staining results of corneal endothelial cells obtained in example 8;
FIG. 13 shows the results of immunofluorescent staining for CD56 of corneal endothelial cells obtained in example 8;
FIG. 14 shows immunofluorescence staining results for Claudin N-cadherin (N-cad) of corneal endothelial cells obtained in example 8;
FIG. 15 is a graph showing the CD56 expression rate as a result of the cell flow of corneal endothelial cells in example 8;
FIG. 16 is a graph showing the expression rate of CD34 as a result of the cell flow of corneal endothelial cells in example 8.
Detailed Description
The invention provides a corneal endothelial cell induction method, which comprises the following steps:
(1) Culturing the induced pluripotent stem cells in an mTeSR1 culture medium, a dual Smad inhibitor culture medium and a first corneal endothelial cell culture medium in sequence to obtain differentiated induced pluripotent stem cells;
(2) Digesting the differentiated induced pluripotent stem cells, and blowing off the cells by using a second corneal endothelial cell culture medium to obtain a cell suspension;
(3) And culturing the cell suspension with a mixed culture medium to obtain the corneal endothelial cells.
In the present invention, the induced pluripotent stem cells are preferably cultured in mTeSR1 medium for 3 to 5 days.
In the present invention, the manufacturer of the mTeSR1 medium is Stem cell technologies.
In the present invention, the producer of the DMEM/F12 medium is Gibco.
In the present invention, the producer of KSR is Gibco.
In the present invention, the manufacturer of the NEAA is Gibco.
In the present invention, the manufacturer of the human recombinant Noggin is R & D systems.
In the present invention, the manufacturer of the SB431542 is selelck.
In the present invention, the producer of said β -mercaptoethanol is Gibco.
In the present invention, the producer of bFGF is Gibco.
In the present invention, the B27 supplement is a 50 × B27 supplement produced by Gibco.
In the present invention, the manufacturer of PDGF-BB is Gibco.
In the present invention, the manufacturer of DKK-2 used is R & D systems.
In the present invention, the induced pluripotent stem cells are preferably cultured in a dual Smad inhibitor medium for 2 to 4 days.
In the invention, the preparation method of the dual Smad inhibitor culture medium is preferably as follows: preparing a basic culture medium according to the volume ratio of 68.9-84.1 parts of DMEM/F12 culture medium, 15-30 parts of KSR and 0.9-1.1 parts of NEAA, and adding human recombinant Noggin, SB431542, glutamine, beta-mercaptoethanol and bFGF into the basic culture medium to obtain the basic culture medium containing 300-500 ng/mL of human recombinant Noggin, 9-11 mu M SB431542, 0.9-1.1 mM of glutamine, 0.09-0.11 mM of beta-mercaptoethanol and 5-20 ng/mL of bFGF, namely the dual Smad inhibitor culture medium.
In the present invention, the dual Smad inhibitor medium is preferably 70 to 80 parts, and more preferably 75 parts of DMEM/F12 medium.
In the present invention, KSR is preferably 20 to 25 parts, and more preferably 22 to 23 parts in the dual Smad inhibitor medium.
In the present invention, the NEAA in the dual Smad inhibitor medium is preferably 1 part.
In the invention, the concentration of the human recombinant Noggin in the dual Smad inhibitor culture medium is preferably 350-450 ng/mL, and more preferably 400ng/mL.
In the present invention, the concentration of SB431542 in the dual Smad inhibitor medium is preferably 10 μ M.
In the present invention, the glutamine concentration in the dual Smad inhibitor medium is preferably 10 μ M.
In the present invention, the concentration of β -mercaptoethanol in the dual Smad inhibitor medium is preferably 0.1mM.
In the invention, the bFGF concentration in the dual Smad inhibitor culture medium is preferably 10-15 ng/mL.
In the present invention, the induced pluripotent stem cells are preferably cultured in the first corneal endothelial cell culture medium for 1 to 3 days.
In the present invention, the first corneal endothelial cell culture medium is preferably prepared by: preparing a basal culture medium according to the volume ratio of 68.9-84.1 parts of DMEM/F12 culture medium, 15-30 parts of KSR and 0.9-1.1 parts of NEAA, and adding B27 supplement, PDGF-BB, DKK-2, L-glutamine, beta-mercaptoethanol and bFGF into the basal culture medium to obtain the basal culture medium containing 0.1 XB 27 supplement, 9-11 ng/mL PDGF-BB, 9-11 ng/mL DKK-2, 0.8-1.2 mM L-glutamine, 0.09-0.11 mM beta-mercaptoethanol and 5-20 ng/mLbFGF, namely the first corneal endothelial cell culture medium.
In the present invention, the first corneal endothelial cell culture medium is preferably 70 to 80 parts by weight of DMEM/F12, and more preferably 75 parts by weight.
In the present invention, the KSR is preferably 20 to 25 parts, and more preferably 22 to 23 parts, in the first corneal endothelial cell culture medium.
In the present invention, the first corneal endothelial cell culture medium preferably contains 1 part of NEAA.
In the present invention, the PDGF-BB concentration in the first corneal endothelial cell culture medium is preferably 10ng/mL.
In the present invention, the concentration of DKK-2 in the first corneal endothelial cell culture medium is preferably 10ng/mL.
In the present invention, the concentration of L-glutamine in the first corneal endothelial cell culture medium is preferably 0.9 to 1.1mM, and more preferably 1mM.
In the present invention, the concentration of β -mercaptoethanol in the first corneal endothelial cell culture medium is preferably 0.1mM.
In the present invention, the bFGF concentration in the first corneal endothelial cell culture medium is preferably 10 to 15ng/mL.
In the present invention, the concentration of the B27 supplement in the first corneal endothelial cell culture medium is preferably 0.1 xb 27 supplement.
In the present invention, it is preferable to digest with Accutase during the digestion process.
In the present invention, the digestion temperature is preferably 35 to 39 ℃, more preferably 36 to 38 ℃, and still more preferably 37 ℃.
In the present invention, the digestion time is preferably 3 to 7min, more preferably 4 to 6min, and still more preferably 5min.
In the present invention, it is preferable that the digested liquid is removed before the second corneal endothelial cell culture medium is added to blow off the cells.
In the present invention, the mixed culture medium is preferably prepared from a second corneal endothelial cell culture medium and a corneal endothelial cell conditioned medium in a volume ratio of 1:0.8 to 1.2, and more preferably 1:1 in the ratio of (1).
In the present invention, the culture time in the mixed culture medium is preferably 6 to 8 days, and more preferably 7 days.
In the present invention, the volume ratio of the cell suspension and the mixed culture medium in the step (3) is preferably 1:1.8 to 2.2, more preferably 1:1.9 to 2.1, and more preferably still 1:2.
in the present invention, the second corneal endothelial cell culture medium is preferably prepared by a method comprising: preparing a basic culture medium according to the volume ratio of DMEM/F1245-47 parts, M19945-47 parts, FBS 4.5-5.5 parts, NEAA 0.8-1.2 parts, glutaMAX 0.8-1.2 parts and ITS 0.8-1.2 parts, adding bFGF and penicillin-streptomycin into the basic culture medium to obtain the basic culture medium containing 5-20 ng/mLbFGF and 95-105 units/ml penicillin-streptomycin, namely the second corneal endothelial cell culture medium.
In the present invention, the DMEM/F12 in the second corneal endothelial cell culture medium is preferably 46 parts.
In the present invention, M199 in the second corneal endothelial cell culture medium is preferably 46 parts.
In the present invention, FBS in the second corneal endothelial cell culture medium is preferably 5 parts.
In the present invention, the NEAA in the second corneal endothelial cell culture medium is preferably 0.9 to 1.1 parts, and more preferably 1 part.
In the present invention, the second corneal endothelial cell culture medium preferably contains 0.9 to 1.1 parts of GlutaMAX, and more preferably contains 1 part of GlutaMAX.
In the present invention, the ITS in the second corneal endothelial cell culture medium is preferably 0.9 to 1.1 parts, and more preferably 1 part.
In the present invention, the bFGF concentration in the second corneal endothelial cell culture medium is preferably 10 to 15ng/mL.
In the present invention, the concentration of penicillin-streptomycin in the second corneal endothelial cell culture medium is preferably 100units/ml.
In the present invention, the preparation method of the corneal endothelial cell conditioned medium is preferably: digesting the elastic layer behind the cornea to obtain a corneal endothelial cell cluster, continuously digesting to obtain a single cell, culturing to be fused by using a second corneal endothelial culture medium, subculturing to a P3 generation, and collecting culture supernatant of the P1-P3 generation, namely the corneal endothelial cell conditioned medium.
In the present invention, collagenase is preferably used for digestion of the elastic layer after the cornea.
In the present invention, the collagenase concentration is preferably 1 to 3mg/ml, and more preferably 2mg/ml.
In the present invention, the temperature at which the elastic layer is digested after the cornea is preferably 35 to 39 ℃, and more preferably 37 ℃.
In the present invention, the digestion time of the retrocorneal elastic layer is preferably 2 to 4 hours, and more preferably 3 hours.
In the present invention, it is preferable to digest the corneal endothelial cell clusters by using pancreatic enzyme.
In the present invention, the digestion time of the corneal endothelial cell clusters is preferably 1 to 3min, and more preferably 2min.
In the present invention, the cells are preferably digested with pancreatin for 3 to 7min, and more preferably digested with pancreatin for 5min before subculture.
In the present invention, the pancreatin is preferably 0.2 to 0.3% pancreatin, and more preferably 0.25% pancreatin.
In the present invention, the pancreatic enzyme is preferably used at a temperature of 36 to 38 ℃ and more preferably at a temperature of 37 ℃.
In the present invention, the cell suspension is preferably inoculated into a Matrigel-coated 6-well plate and then mixed with a mixed medium.
In the invention, the induced corneal endothelial cells can obtain high-purity uniform corneal endothelial cells by means of continuous passage.
In the present invention, the passaging density is preferably 10000 to 30000 cells/cm 2 More preferably 15000 to 25000 cells/cm 2 Still more preferably 20000 cells/cm 2 。
In the present invention, the total content of CO is preferably 36 to 38 ℃ and 4 to 6% 2 Under conditions of, more preferably, 37 ℃,5% 2 Under the conditions of the reaction.
In the present invention, the culture medium was changed every day during all the cultivation.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
(1) Preparing a culture medium: preparing a basal culture medium according to the volume ratio of 68.9 parts of DMEM/F12 culture medium, 30 parts of KSR and 1.1 parts of NEAA, and adding human recombinant Noggin, SB431542, glutamine, beta-mercaptoethanol and bFGF into the basal culture medium to obtain a basal culture medium containing 300ng/mL human recombinant Noggin, 11 mu M SB431542, 1.1mM glutamine, 0.11mM beta-mercaptoethanol and 20ng/mL bFGF, namely the dual Smad inhibitor culture medium;
preparing a basal culture medium according to the volume ratio of 68.9 parts of DMEM/F12 culture medium, 30 parts of KSR and 1.1 parts of NEAA, adding a B27 supplement, PDGF-BB, DKK-2, L-glutamine, beta-mercaptoethanol and bFGF into the basal culture medium to obtain a basal culture medium containing 0.09 XB 27 supplement, 11 ng/mLPDGGF-BB, 11ng/mLDKK-2, 1.2mM L-glutamine, 0.11mM beta-mercaptoethanol and 20ng/mL bFGF, namely a first corneal endothelial cell culture medium;
preparing a basic culture medium according to the volume ratio of DMEM/F1245 parts, M19947 parts, FBS4.5 parts, NEAA 0.8 parts, glutaMAX0.8 parts and ITS 0.8 parts, adding bFGF and penillin-streptomycin into the basic culture medium to obtain a basic culture medium containing 5ng/mL bFGF and 95units/mL penillin-streptomycin, namely a second corneal endothelial cell culture medium
Digesting the elastic layer behind the cornea for 4 hours at 39 ℃ by using 1mg/ml collagenase to obtain a corneal endothelial cell cluster, continuously digesting for 1min at 36 ℃ by using 0.3% pancreatin to obtain a single cell, culturing by using a second corneal endothelial culture medium until the single cell is fused, digesting for 3min at 36 ℃ by using 0.3% pancreatin, subculturing to P3 generation, and collecting culture supernatant of the P1-P3 generation, namely the corneal endothelial cell conditioned medium.
(2) Culturing the induced pluripotent stem cells in an mTeSR1 culture medium for 3 days, replacing the mTeSR1 culture medium with a dual Smad inhibitor culture medium for 2 days, and replacing the dual Smad inhibitor culture medium with a first corneal endothelial cell culture medium for 1 day to obtain differentiated induced pluripotent stem cells;
(3) Digesting the differentiated induced pluripotent stem cells for 7min by using Accutase at 35 ℃, discarding a digestion solution, and blowing off the cells by using a second corneal endothelial cell culture medium to obtain a cell suspension;
(4) And (3) mixing the second corneal endothelial cell culture medium and the corneal endothelial cell conditioned medium in a volume ratio of 1:0.8, inoculating the cell suspension into a Matrigel coated 6-well plate, and mixing the cell suspension and the mixed culture medium according to the volume ratio of 1:1.8, adding the mixed culture medium, and culturing for 6 days to obtain the corneal endothelial cells.
Example 2
(1) Preparation of a culture medium: preparing a basal culture medium according to the volume ratio of 74 parts of DMEM/F12 culture medium, 25 parts of KSR and 1 part of NEAA, and adding human recombinant Noggin, SB431542, glutamine, beta-mercaptoethanol and bFGF into the basal culture medium to obtain a basal culture medium containing 400ng/mL human recombinant Noggin, 10 mu M SB431542, 10mM glutamine, 0.1mM beta-mercaptoethanol and 15ng/mL bFGF, namely the dual Smad inhibitor culture medium;
preparing a basal culture medium according to the volume ratio of 79 parts of DMEM/F12 culture medium, 20 parts of KSR and 1 part of NEAA, and adding a B27 supplement, PDGF-BB, DKK-2, L-glutamine, beta-mercaptoethanol and bFGF into the basal culture medium to obtain a basal culture medium containing 0.1 XB 27 supplement,10 ng/mLPDGGF-BB, 10ng/mLDKK-2, 1mM L-glutamine, 0.1mM beta-mercaptoethanol and 10ng/mL bFGF, namely a first corneal endothelial cell culture medium;
preparing a basal culture medium according to the volume ratio of DMEM/F1246 parts, M19946 parts, FBS 5 parts, NEAA1 parts, glutaMAX1 parts and ITS 1 parts, adding bFGF and penicillin-streptomycin into the basal culture medium to obtain a basal culture medium containing 15ng/mL bFGF and 100units/mL penicillin-streptomycin, namely a second corneal endothelial cell culture medium
Digesting the elastic layer behind the cornea for 3 hours at 37 ℃ by using 2mg/ml collagenase to obtain a corneal endothelial cell cluster, continuously digesting for 3 minutes at 37 ℃ by using 0.25% pancreatin to obtain a single cell, culturing by using a second corneal endothelial culture medium until the single cell is fused, digesting for 5 minutes at 37 ℃ by using 0.25% pancreatin, subculturing to P3 generation, and collecting culture supernatant of the P1-P3 generation, namely the corneal endothelial cell conditioned medium.
(2) Culturing the induced pluripotent stem cells in an mTeSR1 culture medium for 4 days, replacing the mTeSR1 culture medium with a dual Smad inhibitor culture medium for 3 days, and replacing the dual Smad inhibitor culture medium with a first corneal endothelial cell culture medium for 2 days to obtain differentiated induced pluripotent stem cells;
(3) Digesting the differentiated induced pluripotent stem cells for 5min by using Accutase at 37 ℃, removing a digestive juice, and blowing off the cells by using a second corneal endothelial cell culture medium to obtain a cell suspension;
(4) And (3) mixing the second corneal endothelial cell culture medium and the corneal endothelial cell conditioned medium in a volume ratio of 1:1, inoculating the cell suspension into a Matrigel coated 6-well plate, and mixing the cell suspension and the mixed culture medium according to the volume ratio of 1:2, and culturing for 7 days to obtain the corneal endothelial cells.
Example 3
(1) Preparing a culture medium: preparing a basic culture medium according to the volume ratio of 84.1 parts of DMEM/F12 culture medium, 15 parts of KSR and 0.9 part of NEAA, and adding human recombinant Noggin, SB431542, glutamine, beta-mercaptoethanol and bFGF into the basic culture medium to obtain a basic culture medium containing 500ng/mL human recombinant Noggin, 9 mu M SB431542, 0.9mM glutamine, 0.09mM beta-mercaptoethanol and 5ng/mL bFGF, namely the dual Smad inhibitor culture medium;
preparing a basal medium according to the volume ratio of 84.1 parts of DMEM/F12 medium, 15 parts of KSR and 0.9 part of NEAA, adding a B27 supplement, PDGF-BB, DKK-2, L-glutamine, beta-mercaptoethanol and bFGF into the basal medium to obtain a basal medium containing 0.11 XB 27 supplement, 9 ng/mLPDGGF-BB, 9ng/mLDKK-2, 0.8mM L-glutamine, 0.09mM beta-mercaptoethanol and 5ng/mLbFGF, namely the first corneal endothelial cell culture medium;
preparing a basal medium according to the volume ratio of DMEM/F1247 parts, M19945 parts, FBS 5.5 parts, NEAA 1.2 parts, glutaMAX1.2 parts and ITS 1.2 parts, adding bFGF and penillin-streptomycin into the basal medium to obtain the basal medium containing 20ng/mL bFGF and 105units/mL penillin-streptomycin, namely the second corneal endothelial cell culture medium
Digesting the elastic layer behind the cornea for 2 hours at 35 ℃ by using collagenase with the concentration of 3mg/ml to obtain corneal endothelial cell clusters, continuously digesting the corneal endothelial cell clusters for 3 minutes at 38 ℃ by using pancreatin with the concentration of 0.2% to obtain single cells, culturing the corneal endothelial cell clusters to be fused by using a second corneal endothelial culture medium, digesting the corneal endothelial cell clusters for 7 minutes at 38 ℃ by using pancreatin with the concentration of 0.2% to perform subculture to a generation P3, and collecting culture supernatants of the generation P1-P3 to obtain the corneal endothelial cell conditioned medium.
(2) Culturing the induced pluripotent stem cells in an mTeSR1 culture medium for 5 days, replacing the mTeSR1 culture medium with a dual Smad inhibitor culture medium for 4 days, and replacing the dual Smad inhibitor culture medium with a first corneal endothelial cell culture medium for 3 days to obtain differentiated induced pluripotent stem cells;
(3) Digesting the differentiated induced pluripotent stem cells for 3min by using Accutase at 39 ℃, discarding a digestion solution, and blowing off the cells by using a second corneal endothelial cell culture medium to obtain a cell suspension;
(4) And (3) mixing the second corneal endothelial cell culture medium and the corneal endothelial cell conditioned medium in a volume ratio of 1:1.2, inoculating the cell suspension into a Matrigel coated 6-well plate, and mixing the cell suspension and the mixed culture medium according to the volume ratio of 1:2.2, adding the mixed culture medium, and culturing for 8 days to obtain the corneal endothelial cells.
Example 4
The corneal endothelial cells induced in example 2 were collected at 20000 cells/cm 2 The passage density of (2) was as shown in FIG. 4, and the passage density of P3 was as shown in FIG. 5. Therefore, the obtained corneal endothelial cells are in hexagonal close connection, and the cells are uniform in high purity.
Example 5
(1) The same medium as in example 2 was prepared;
(2) Culturing the induced pluripotent stem cells in an mTeSR1 culture medium for 4 days, replacing the mTeSR1 culture medium with a dual Smad inhibitor culture medium for 3 days, and replacing the dual Smad inhibitor culture medium with a first corneal endothelial cell culture medium for 2 days to obtain differentiated induced pluripotent stem cells;
(3) Digesting the differentiated induced pluripotent stem cells for 5min at 37 ℃ by using Accutase, removing a digestive juice, and blowing off the cells by using a first corneal endothelial cell culture medium to obtain a cell suspension;
(4) The cell suspension was seeded into Matrigel coated 6-well plates, in a volume ratio of cell suspension to first corneal endothelial cell culture medium of 1:2, the corneal endothelial cells were obtained after culturing for 7 days with the first corneal endothelial cell culture medium added, and as shown in fig. 6, the cells were poorly shaped and unable to proliferate after subculturing to P2 generation, as shown in fig. 7.
Example 6
(1) The same medium as in example 2 was prepared;
(2) Culturing the induced pluripotent stem cells in an mTeSR1 culture medium for 4 days, replacing the mTeSR1 culture medium with a dual Smad inhibitor culture medium for 3 days, and replacing the dual Smad inhibitor culture medium with a first corneal endothelial cell culture medium for 2 days to obtain differentiated induced pluripotent stem cells;
(3) Digesting the differentiated induced pluripotent stem cells for 5min by using Accutase at 37 ℃, removing a digestive juice, and blowing off the cells by using a second corneal endothelial cell culture medium to obtain a cell suspension;
(4) Inoculating the cell suspension into a Matrigel-coated 6-well plate, and mixing the cell suspension and the second corneal endothelial cell culture medium according to the volume ratio of 1:2, and culturing for 7 days to obtain corneal endothelial cells, and the results are shown in fig. 8, and when subcultured to P3 generation, the cell morphology is not very uniform and purification cannot be performed, as shown in fig. 9.
Example 7
(1) Preparing an N2 culture medium: DMEM/F12 (1), 1% N2 supplement,10ng/mlbFGF,1 XNEAA, 2mM Gibco, 100units/mlpenicilin-streptomycin, 2ng/ml human recombinant insulin;
(2) Culturing induced pluripotent stem cells in mTeSR1 culture medium for 4 days, digesting with Accutase for 2min, removing digestive juice, scraping cell clones, inoculating to a low-adsorption 6-pore plate, and performing suspension culture to form embryoid bodies;
(3) The next day, using N2 medium +500ng/ml Noggin +10 μm SB31542 for 4 days;
(4) Inoculating the embryoid bodies to a 6-well plate coated by Matrigel for adherent culture, culturing by using a second corneal endothelial cell culture medium, changing liquid once every 2 days, carrying out passage when the cells have typical morphology, and culturing according to 20000 cells/cm 2 Transmitting to P1, and continuing to culture with a second corneal endothelial cell culture medium;
after the embryoid body is cultured in an adherent way, a large number of fibroblasts and epithelial cells exist, endothelial cells are few, the purification effect is not obvious through passage, and the result is shown in figure 10, and a large number of hybrid cells still exist.
Example 8 identification of corneal endothelial cells induced
1. Immunostaining the corneal endothelial cells obtained by induction
The specific operation is as follows:
(1) The corneal endothelial cells prepared in example 2 were seeded into 12-well plates, respectively;
(2) After the cells form close connection and have a hexagonal typical shape, performing a dyeing step;
(3) Corneal endothelial cells were fixed with 4% paraformaldehyde in PBS buffer at 25 ℃ for 30 minutes;
(4) Permeabilization of the cells with 0.5% triton X-100 in PBS for 10 min;
(5) PBS is washed for three times, 5 minutes each time;
(6) Blocking 6% sheep serum for 1.5 hours, and washing twice with PBS;
(7) Adding primary antibodies respectively: sodium-potassium-atpase (1;
(8) PBS washing for 5 minutes for three times;
(9) Adding secondary antibodies labeled with Cy2 and Cy3, and incubating for 40 minutes at room temperature;
(10) PBS washing for 5 minutes for four times;
(11) Add 0.5. Mu.g/mL DAPI in PBS to stain for 10 min;
(12) Washing with PBS for three times to remove excessive DAPI;
(13) Add 20. Mu.l of mounting medium to mount.
The results of immunofluorescent staining are shown in FIGS. 11-14, showing: the obtained markers of the corneal endothelial cells, namely sodium-potassium-ATPase, CD56, N-cadherin (N-cad) and zon-1, are positively expressed by immunostaining.
2. Flow results of corneal endothelial cells:
the specific operation is as follows:
(1) Digesting the corneal endothelial cells prepared in the example 2 by using Accutase, digesting the corneal endothelial cells for 5min at 37 ℃, collecting and counting the cells after the cells become round and fall off, and centrifuging the cells for 5min at 400 g;
(2) Removing supernatant, resuspending cell pellets by PBS, and centrifuging for 5 minutes at 400 g;
(3) Removing supernatant, resuspending the cell pellet in 4% paraformaldehyde-containing PBS buffer solution, and fixing at room temperature for 15 min;
(4) Washing with PBS, and subpackaging;
(5) Primary anti-incubation: respectively adding 3 mul of Isotype control and a proper amount of anti-CD 56/CD34, and incubating for 1.5 hours at 4 ℃ in the dark;
(6) Directly adding PBS into a tube with the volume of 1mL, uniformly mixing, and centrifuging for 5 minutes at 400 g;
(7) Then adding a secondary antibody, and incubating for 30 minutes at room temperature;
(8) Adding 1ml PBS for cleaning, eluting for 2 times, and adding PBS for resuspension;
(9) And (5) detecting by an up-flow instrument.
The corneal endothelial cell flow results are shown in fig. 15 to 16, in which the expression rate of CD56 was 91.4% and the expression rate of CD34 was 4.5%.
As can be seen from the above examples, the present invention provides a method for inducing corneal endothelial cells, comprising the steps of (1) culturing induced pluripotent stem cells in an mTeSR1 medium, a dual Smad inhibitor medium, and a first corneal endothelial cell medium in this order to obtain differentiated induced pluripotent stem cells; (2) Digesting the differentiated induced pluripotent stem cells, and blowing off the cells by using a second corneal endothelial cell culture medium to obtain a cell suspension; (3) And culturing the cell suspension with a mixed culture medium to obtain the corneal endothelial cells. The induction method of the invention can obtain a large amount of high-purity corneal endothelial cells in a short time, and the obtained corneal endothelial cells can be passaged in vitro for at least 4 times, thereby effectively solving the problems of limited number of human corneal donors and limited in vitro amplification capacity of primary corneal endothelial cells.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.
Claims (1)
1. A method for inducing corneal endothelial cells, comprising the steps of:
(1) Culturing the induced pluripotent stem cells in an mTeSR1 culture medium, a dual Smad inhibitor culture medium and a first corneal endothelial cell culture medium in sequence to obtain differentiated induced pluripotent stem cells;
(2) Digesting the differentiated induced pluripotent stem cells, and blowing off the cells by using a second corneal endothelial cell culture medium to obtain a cell suspension;
(3) Culturing the cell suspension with a mixed culture medium to obtain corneal endothelial cells;
the induced pluripotent stem cells are cultured in an mTeSR1 culture medium for 3-5 days;
the induced pluripotent stem cells are cultured in a dual Smad inhibitor culture medium for 2-4 days, and the preparation method of the dual Smad inhibitor culture medium comprises the following steps: preparing a basic culture medium according to the volume ratio of 68.9-84.1 parts of DMEM/F12 culture medium, 15-30 parts of KSR and 0.9-1.1 parts of NEAA, and adding human recombinant Noggin, SB431542, glutamine, beta-mercaptoethanol and bFGF into the basic culture medium to obtain the basic culture medium containing 300-500 ng/mL human recombinant Noggin, 9-11 mu M SB431542, 0.9-1.1 mM glutamine, 0.09-0.11 mM beta-mercaptoethanol and 5-20 ng/mLbFGF, namely the dual Smad inhibitor culture medium;
the time for culturing the induced pluripotent stem cells in the first corneal endothelial cell culture medium is 1-3 days, and the preparation method of the first corneal endothelial cell culture medium comprises the following steps: preparing a basal culture medium according to the volume ratio of 68.9-84.1 parts of DMEM/F12 culture medium, 15-30 parts of KSR and 0.9-1.1 parts of NEAA, adding a B27 supplement, PDGF-BB, DKK-2, L-glutamine, beta-mercaptoethanol and bFGF into the basal culture medium to obtain the basal culture medium containing 0.09-0.11 XB 27 supplement, 9-11 ng/mLPDGF-BB, 9-11 ng/mL DKK-2, 0.8-1.2 mM L-glutamine, 0.09-0.11 mM beta-mercaptoethanol and 5-20 ng/mLbFGF, namely the first corneal endothelial cell culture medium;
during the digestion process, accutase is used for digestion, the digestion temperature is 35-39 ℃, and the digestion time is 3-7 min;
removing digested digestive juice during cell blowing, and adding a second corneal endothelial cell culture medium;
the mixed culture medium is prepared by mixing a second corneal endothelial cell culture medium and a corneal endothelial cell conditioned medium according to the volume ratio of 1:0.8 to 1.2, and the culture time in the mixed culture medium is 6 to 8 days;
the volume ratio of the cell suspension to the mixed culture medium in the step (3) is 1:1.8 to 2.2;
the preparation method of the second corneal endothelial cell culture medium comprises the following steps: preparing a basic culture medium according to the volume ratio of DMEM/F12-47 parts, M199-47 parts, FBS 4.5-5.5 parts, NEAA 0.8-1.2 parts, glutaMAX 0.8-1.2 parts and ITS 0.8-1.2 parts, adding bFGF and penicillin-streptomycin into the basic culture medium to obtain a basic culture medium containing 5-20 ng/mL bFGF and 95-105 units/mL penicillin-streptomycin, namely a second corneal endothelial cell culture medium;
the preparation method of the corneal endothelial cell conditioned medium comprises the following steps: digesting the elastic layer behind the cornea to obtain a corneal endothelial cell cluster, continuously digesting to obtain a single cell, culturing to be fused by using a second corneal endothelial culture medium, subculturing to a P3 generation, and collecting culture supernatant of the P1-P3 generation, namely the corneal endothelial cell conditioned medium.
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