CN108384746B - Method for efficiently differentiating induced pluripotent stem cells into mature endothelial cells - Google Patents
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Abstract
The invention discloses a method for efficiently differentiating induced pluripotent stem cells into mature endothelial cells, belonging to the technical field of cell engineering. Which comprises the following steps: inducing pluripotent stem cells to perform pretreatment culture; inducing formation of lateral plate mesoderm cells; inducing the formation of endothelial progenitor cells that are predisposed to endothelial cells; inducing the formation of mature endothelial cells. The invention can efficiently induce pluripotent stem cells to directionally differentiate towards endothelial cells by increasing, decreasing and matching various cell induction factors and reasonably upgrading the induction method, and can obtain a large amount of mature endothelial cells with high purity and complete functions under the conditions of adopting a small amount of induced pluripotent stem cells and not needing purification operation.
Description
Technical Field
The invention relates to a method for efficiently differentiating induced pluripotent stem cells into mature endothelial cells, belonging to the technical field of cell engineering.
Background
Vascular Endothelial Cells (vecs) are a layer of specific squamous epithelial Cells that form the inner wall of blood vessels, and are an important guarantee for maintaining the stable state of the systemic blood circulation system. In diabetes, hypertension and other chronic diseases, endothelial cell dysfunction or damage is often accompanied, cardiovascular diseases such as atherosclerosis and even myocardial infarction are easily induced, and the life health of patients is seriously threatened. Therefore, modulation of repair or replacement of damaged vascular endothelium has important clinical value for prevention and treatment of cardiovascular diseases. Researches indicate that mature vascular endothelial cells with complete functions can reform vascular tissues in the area of the vascular infarction of a human body, and the ischemic symptom is effectively solved. In addition, the vascular structure formed by mature vascular endothelial cells in vitro has great potential to replace the conventional vein separation means, and provides a substitute material with stronger applicability for the heart bypass surgery of patients with cardiovascular diseases. However, how to obtain a large amount of mature vascular endothelial cells with the capability of tube formation is still in the research and exploration stage at present, which directly limits the successful development of the mature vascular endothelial cells in clinical application.
The emergence of induced pluripotent stem Cells (ipscs), which is an important factor in the development of regenerative medicine, provides a highly feasible approach for the clinical application of Endothelial Cells (ECs). The induced pluripotent stem cells successfully bypass two most key problems of immunogenicity and ethics, are wide in source, and can complete individualized treatment by establishing patient-specific induced pluripotent stem cells. The induced pluripotent stem cells from patients are adopted to induce and expand in vitro to obtain a large number of mature vascular endothelial cells, which is beneficial to smoothly crossing the high threshold of EC clinical application and can also prepare for future gene therapy aiming at the endothelial cells.
In recent years, the related research fields are rapidly developed, and various methods for inducing the differentiation of pluripotent stem cells into mature endothelial cells are successively proposed, including the early 3D embryoid body induction method[1]And recent 2D direct induction method[2-4]A series of induction factors required by endothelial differentiation are adopted for multiple combination, and the differentiation of the induced pluripotent stem cells to mature endothelial cells can be induced to a certain extent. However, these induction methods also have limitations, such as long induction period (generally requiring 9-20 days), low transformation efficiency (20% -60%), complicated purification process (requiring screening by MACS or flow), incomplete endothelial function (lacking in vitro or in vivo tube-forming ability), and the like.
In view of this, in order to obtain endothelial cells of a clinical application level, further research and optimization of a method for inducing differentiation of pluripotent stem cells into endothelial cells is required.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for efficiently differentiating induced pluripotent stem cells into mature endothelial cells. The invention can efficiently induce pluripotent stem cells to directionally differentiate towards endothelial cells by increasing, decreasing and matching various cell induction factors and reasonably upgrading the induction method, and can obtain a large amount of mature endothelial cells with high purity and complete functions under the conditions of adopting a small amount of induced pluripotent stem cells and not needing purification operation. Meanwhile, the whole induction process is short in time consumption and simple to operate, animal-derived cells and components are not involved, the high efficiency, the functionality and the safety of the endothelial cells derived from the induced pluripotent stem cells are greatly improved, and a new way and a new method are provided for producing mature endothelial cells at the clinical application level on a large scale by utilizing the induced pluripotent stem cells of a patient.
The technical scheme for solving the technical problems is as follows: a method for efficiently differentiating induced pluripotent stem cells into mature endothelial cells comprises the following steps:
step 1: pretreating induced pluripotent stem cells to obtain cell resuspension;
step 2: the cell resuspension obtained in step 1 is added according to the proportion of each 1cm2The bottom area of the cell culture plate is paved with 2-5 ten thousand cells, the cells are transferred into a cell culture plate containing a mixture of TeSR-E8 culture medium and ROCK inhibitor Y27632, and the cell culture plate is placed in a cell culture box for culturing for 16-24 hours;
and step 3: removing the old medium from the cell culture plate in step 2 at a rate of every 10cm2The liquid adding amount of the bottom area of the cell culture plate is 4mL, a first N2B27 insulin deficiency culture medium is added, and the cell culture plate is placed in a cell culture box for culture for 16 to 24 hours;
and 4, step 4: removing the old medium from the cell culture plate in step 3, and washing the cell culture plate with PBS every 10cm2Adding a second N2B27 insulin deletion culture medium into the cell culture plate at a ratio of 6mL of liquid adding amount of the bottom area of the cell culture plate, placing the cell culture plate in a cell culture box for culturing for 48 hours, and obtaining lateral plate mesoderm cells in the cell culture plate;
and 5: the old medium in the cell culture plate containing the side plate mesoderm cells in step 4 was removed, and the cell culture plate was washed with PBS at every 10cm2The liquid adding amount of the bottom area of the cell culture plate is 4mL, the first StemPro-34 culture medium is added, then the cell culture plate is placed in a cell culture box for culturing for 16 to 24 hours, the operation of the step 5 is repeated once,inducing endothelial progenitor cells with endothelial cell tendency in a cell culture plate;
step 6: removing the old medium from the cell culture plate containing endothelial progenitor cells that are prone to endothelial cell in step 5, and washing the cell culture plate with PBS at a rate of every 10cm2The liquid adding amount of the cell culture plate bottom area is 0.5mL, and cell-decomposing enzyme mixed liquor is added for enzymolysis to obtain endothelial progenitor cells which are subjected to enzymolysis and have endothelial cell tendency;
and 7: the endothelial progenitor cells which are obtained in the step 6 and have the endothelial cell tendency after enzymolysis are distributed according to the proportion of every 10cm2Adding a second StemPro-34 culture medium into the cell culture plate with the liquid adding amount of 1mL to obtain endothelial progenitor cell resuspension containing endothelial cell tendency;
and 8: re-suspending the endothelial progenitor cell suspension with the endothelial cell tendency obtained in the step 7,
per 1cm2The bottom area of the cell culture plate is paved with 3-6 ten thousand cells, the cells are transferred into a cell differentiation culture plate containing a second StemPro-34 culture medium, and the cells are placed in a cell culture box for culture for 16-24 hours;
and step 9: removing the old medium from the cell culture plate in step 8, and washing the cell culture plate with PBS at a rate of every 10cm2The liquid adding amount of the bottom area of the cell culture plate is 2mL, a second StemPro-34 culture medium is added, and the cell culture plate is placed in a cell culture box and cultured for 48 hours to obtain mature induced differentiation endothelial cells.
Compared with the prior art, the method does not need a purification step, the purity of the obtained mature endothelial cells is up to more than 96 percent, the operation difficulty is reduced, and the interference of the purification step on the cell state is reduced. Taking 6-well cell culture plates as an example, 30-35 ten thousand induced pluripotent stem cells are initially seeded in each well, and 400-500 ten thousand mature endothelial cells can be harvested at the induction day 9, which is 2-3 times the yield of endothelial cells in the prior art. By adopting the method of the invention, the transformation ratio of the obtained endothelial cells/the initial induced pluripotent stem cells can reach 15/1 after 9 days of induction, and is 2-3 times of the transformation ratio of the endothelial cells/the initial induced pluripotent stem cells in the prior art. The mature endothelial cells obtained by the invention have obvious in-vitro tube forming capability.
Wherein, in the step 3, the old culture medium in the cell culture plate in the step 2 is removed, and the specific operation method comprises the following steps: the plate was tilted and old medium was aspirated with a pipette.
The method of removing old medium from the cell culture plate in step 4, step 5, step 6, step 9 is the same as the method of removing old medium from the cell culture plate in step 3 described above.
In step 5, Endothelial progenitor cells, i.e., Endothelial progenitor cells, EPC for short.
On the basis of the technical scheme, the invention can be further improved as follows.
Further, in step 1, the method for pretreating induced pluripotent stem cells to obtain a cell resuspension comprises:
in a cell culture plate, after the induced pluripotent stem cells are recovered and passaged, the induced pluripotent stem cells are washed for 2 times by PBS (phosphate buffer solution), and then the concentration of the induced pluripotent stem cells is 10cm per minute2Adding 0.5mL of liquid into the bottom area of the cell culture plate, and adding a cell digestive juice preheated at 37 ℃ for enzymolysis digestion to obtain an enzymolysis digestive juice; when 70-80% of induced pluripotent stem cells begin to fall off, adding DMEM/F12 culture medium with the volume 5 times that of the enzymolysis digestion solution for neutralization, then transferring the induced pluripotent stem cells into a centrifuge tube, centrifuging, removing supernatant, adding TeSR-E8 culture medium, and carrying out heavy suspension on the cell sediment to obtain cell heavy suspension.
The DMEM/F12 medium was purchased from Life Technologies, Inc. and was designated 11039021.
Furthermore, the method for recovering and passaging the induced pluripotent stem cells comprises the following steps:
preparing a cell culture plate coated by a basement membrane matrix one day before recovery, placing the cell culture plate in a cell culture box, and incubating overnight to obtain the overnight incubated cell culture plate coated by the basement membrane matrix;
taking out the cryopreserved induced pluripotent stem cells from liquid nitrogen, inoculating the cells into the cell culture plate coated by the basement membrane substrate after overnight incubation, placing the cells into a cell culture box, and incubating for 16-24 hours;
then replacing a fresh TeSR-E8 culture medium preheated at 37 ℃, and repeatedly changing the culture medium every day until the cell polymerization degree is increased to 75-85%, thus obtaining the recovered induced pluripotent stem cells;
and (3) carrying out passage on the recovered induced pluripotent stem cells, replacing a fresh TeSR-E8 culture medium preheated at 37 ℃ after one day, and repeatedly replacing the culture medium every day until the cell polymerization degree is increased to 75-85%, thus obtaining the induced pluripotent stem cells after passage.
Further, the centrifugation conditions were room temperature, 200g, and 5 min.
Further, in step 2, the concentration of ROCK inhibitor Y27632 in the mixture of TeSR-E8 medium and ROCK inhibitor Y27632 was 10. mu.M/L.
Further, in step 3, the first N2B27 insulin deficiency medium is obtained by adding BMP4, CHIR-99021 and Activin A to N2B27 insulin deficiency medium, wherein the concentration of BMP4 is 20-30ng/mL, the concentration of CHIR-99021 is 6-10 μ M/L, and the concentration of Activin A is 30-70 ng/mL.
BMP4, CHIR-99021 and Activin A are small inducing molecules, wherein BMP4, which is called BoneMorphogenic Protein4 in its entirety, is named bone morphogenetic Protein4, which has a direct or indirect relationship with cell development such as cell proliferation and differentiation, apoptosis, cell signaling during morphogenesis, and the like, CHIR-99021 is a GSK inhibitor, which inhibits GSK3 β and GSK3 α with high specificity, Activin A is Activin A, which is a pleiotropic cytokine involved in various important biological processes, including the maintenance of pluripotent stem cells.
The adoption of the further beneficial effects is as follows: BMP4 and CHIR-99021 can start the induced differentiation process from induced pluripotent stem cells to lateral plate mesoderm cells, and Activin A can further promote the differentiation process of induced pluripotent stem cells on the basis, so that the final endothelial transformation efficiency is improved. Since Activin a treatment time is too long, which results in the induced pluripotent stem cells eventually differentiating into endoderm cells, whereas endothelial cells are derived from mesoderm cells only, Activin a treatment time needs to be controlled within 24 hours.
Further, the concentration of BMP4, CHIR-99021 and Activin A in the first N2B27 insulin deficiency medium was 25ng/mL, 8. mu.M/L and 50ng/mL, respectively.
Further, in step 4, the second N2B27 insulin deficiency medium is obtained by adding BMP4 and CHIR-99021 to N2B27 insulin deficiency medium, wherein the concentration of BMP4 is 20-30ng/mL, and the concentration of CHIR-99021 is 6-10. mu.M/L.
The adoption of the further beneficial effects is as follows: BMP4 and CHIR-99021 initiate the induced differentiation process of induced pluripotent stem cells into lateral plate mesoderm cells.
Further, the concentration of BMP4 in the second N2B27 insulin deficiency medium was 25ng/mL and the concentration of CHIR-99021 was 8. mu.M/L.
Further, in step 5, the first StemPro-34 medium is obtained by adding VEGFA, Forskolin and SB431542 to the StemPro-34 medium, wherein the concentration of VEGFA is 100-300ng/mL, the concentration of Forskolin is 1-4. mu.M/L, and the concentration of SB431542 is 5-15. mu.M/L.
The VEGFA, Forskolin and SB431542 are all small inducible molecules, wherein VEGFA, which is called Vascular endothelial growth Factor A, is a vascular endothelial growth Factor with high specificity, and has the functions of promoting vascular permeability increase, extracellular matrix degeneration, vascular endothelial cell migration, proliferation and angiogenesis, Forskolin, which is Forskolin, is an adenylate cyclase activator, SB431542 is a powerful small molecule inhibitor and selectively inhibits transforming growth Factor β (TGF- β), receptor activator I-type kinase ALK5(IC 50: 94nM), ALK4(IC 50: 140nM) and ALK 7.
The adoption of the further beneficial effects is as follows: the combination of VEGFA, Forskolin and SB431542 can efficiently and rapidly guide and induce the pluripotent stem cells to differentiate from mesodermal cells to endothelial cells, and improve the endothelial transformation efficiency.
Further, the concentration of VEGFA in the first StemPro-34 medium was 200ng/mL, the concentration of Forskolin was 2. mu.M/L, and the concentration of SB431542 was 10. mu.M/L.
Further, in step 6, the cell lysis enzyme mixture is composed of cell digest and trypsin at a volume ratio of 1: 1.
Further, in step 7, the second StemPro-34 culture medium is obtained by adding VEGFA to the StemPro-34 culture medium, wherein the concentration of VEGFA is 30-70 ng/mL.
The adoption of the further beneficial effects is as follows: the second stempo-34 medium containing VEGFA can be used to induce further maturation of endothelial cells while also maintaining normal growth proliferation of endothelial cells.
Further, the concentration of VEGFA in the second StemPro-34 medium was 50 ng/mL.
Further, in step 8, the cell differentiation culture plate containing the second stemro-34 medium is obtained by: per 10cm2The amount of the solution added to the bottom area of the cell culture plate is 2mL, 10 mu g/mL of fibronectin working solution is added into the cell culture plate, the cell culture plate is kept stand at room temperature for coating for 1 hour, the fibronectin working solution is removed, and the concentration of the fibronectin working solution per 10cm is determined2The amount of the added liquid on the bottom area of the cell culture plate is 2mL, and a second StemPro-34 culture medium is added, thus obtaining the cell differentiation culture plate containing the second StemPro-34 culture medium.
Further, the culture conditions of the cell culture box are all 37 ℃ and 5% v/v CO2。
Further, step 9 is followed by step 10 of expanding and cryopreserving the obtained mature differentiation-inducing endothelial cells.
The specific method for amplifying the obtained mature induced differentiation endothelial cells comprises the following steps:
when the cell polymerization degree is expanded to 80-90%, the passage operation can be started.
The cell culture plate was removed, the old medium was aspirated, washed 2 times with PBS at room temperature, and then washed every 10cm2The amount of the added liquid in the bottom area of the cell culture plate is 0.5mL, a cell-decomposing enzyme mixed solution which is preheated at 37 ℃ and consists of cell digestive juice and trypsin according to the volume ratio of 1:1 is added, and the cell-decomposing enzyme mixed solution is placed at 37 ℃ and 5% v/v CO2The cell culture box is maintained for 2-3min until most of the cells begin to float and fall off in the form of lumps.
Per 10cm2The liquid adding amount of the cell culture plate bottom area is 1mL, the first StemPro-34 culture medium preheated at 37 ℃ is added to neutralize the enzymolysis of cell digestive juice and trypsin, the exfoliated cell mass is lightly blown by a pipette gun to be resuspended, and then the cell culture plate is transferred to a 15mL centrifuge tube.
At room temperature, 200g, centrifuge for 5 min. The supernatant was removed with a pipette gun, followed by 3mL of a second StemPro-34 medium pre-warmed at 37 ℃ and gently pipetting to resuspend the cell pellet well.
Per 10cm2The amount of the solution added to the bottom area of the cell culture plate was 2mL, and a second StemPro-34 medium preheated to 37 ℃ was added to the cell culture plate. Subsequently, 3mL of the cell resuspension was aliquoted into cell culture plates at 1:3 or 1:2 passages, placed at 37 ℃ in 5% v/v CO2And (4) performing static culture on the cell culture box.
The specific method for cryopreserving the obtained mature induced differentiation endothelial cells comprises the following steps:
observing the cells under a mirror, determining that the EC cells have no pollution and good state, and performing cryopreservation operation on the EC cells when the polymerization degree reaches 80-90%.
The cell culture plate was removed, the old medium was aspirated, washed 2 times with PBS at room temperature, and then washed every 10cm2The amount of the added liquid in the bottom area of the cell culture plate is 0.5mL, a cell-decomposing enzyme mixed solution which is preheated at 37 ℃ and consists of cell digestive juice and trypsin according to the volume ratio of 1:1 is added, and the cell-decomposing enzyme mixed solution is placed at 37 ℃ and 5% v/v CO2The cell culture box is maintained for 2-3min until most of the cells begin to float and fall off in the form of lumps.
Per 10cm2The liquid adding amount of the cell culture plate bottom area is 1mL, a second StemPro-34 culture medium preheated at 37 ℃ is added to neutralize the enzymolysis of cell digestive juice and trypsin, and the exfoliated cell mass is lightly blown by a pipette gun to be resuspended, and then transferred into a 15mL centrifuge tube.
Centrifuge at 200g for 5min at room temperature. During the period, a cryopreservation tube is prepared and marked (name of culture person, cryopreservation time, culture generation number, etc.).
The supernatant was aspirated off with a pipette gun, followed by addition of 2mL of the cryo-SFM stock solution, gentle pipetting to thoroughly resuspend the cell pellet, and then aliquoting into cryotubes at 1mL per tube (a 1:2 ratio is recommended for cryopreservation).
And (3) putting the freezing tube into a program cooling box with pre-cooling at 4 ℃, putting the box into a refrigerator with the temperature of 80 ℃ below zero, and transferring the box into a liquid nitrogen tank for storage the next day.
The specific preparation method of the culture medium used by the invention comprises the following steps:
1. TeSR-E8 medium: the preparation is prepared at present according to the specification proportion shown in Table 1, and is stored for 1 month at the temperature of 4 ℃.
TABLE 1 preparation of TeSR-E8 Medium
2. N2B27 insulin deficiency medium: the preparation is prepared just before use according to the specification proportion shown in Table 2, and is stored for 1 month at the temperature of 4 ℃.
TABLE 2 preparation of N2B27 insulin deficiency Medium
3. StemPro-34 Medium: the preparation is prepared just before use according to the specification proportion shown in Table 3, and is stored for 1 month at the temperature of 4 ℃.
TABLE 3 preparation of StemPro-34 Medium
The invention has the beneficial effects that:
1. the whole induction process of the invention has short time consumption and simple operation, does not relate to animal-derived cells and components, greatly improves the high efficiency, functionality and safety of endothelial cells derived from induced pluripotent stem cells, and provides a new way and method for producing mature endothelial cells at the clinical application level on a large scale by utilizing the induced pluripotent stem cells of patients.
2. The invention can efficiently induce pluripotent stem cells to directionally differentiate towards endothelial cells by increasing, decreasing and matching various cell induction factors and reasonably upgrading the induction method, and can obtain a large amount of mature endothelial cells with high purity and complete functions under the conditions of adopting a small amount of induced pluripotent stem cells and not needing purification operation.
3. The method of the invention also has the following remarkable advantages and positive effects:
(1) short-term property: the method only needs 8-9 days, the time consumption reaches the fastest level in the related field at present, and the method has high application timeliness in clinical application.
(2) Convenience: the method simplifies and optimizes the combination of induction factors, is simple and easy to operate, and has stable repeatability.
(3) High purity: the purity of the cells is extremely high, and mature endothelial cells with the purity of more than 96 percent can be obtained without additional purification steps.
(4) High efficiency: the cell yield is obvious, a large amount of mature endothelial cells can be obtained by adopting a small amount of induced pluripotent stem cells, and the finally obtained endothelial cell/induced pluripotent stem transformation ratio can reach 15/1 within 8-9 days.
(5) Functionality: the obtained mature endothelial cells have obvious tube forming capability and complete functions.
(6) Safety: the induction process is transparent and easy to monitor, and does not relate to animal-derived cells and components; the induction is thorough, no stem cells are left, and the risk of tumor formation is eliminated.
Drawings
FIG. 1 is an overall flow chart of the present invention.
FIG. 2 is a graph showing morphological characteristics of cells at day 1 during induction formation of mesodermal cells in the side plates (10X microscopic field).
FIG. 3 is a graph of the morphological characteristics of cells at day 2 during induction of the formation of mesodermal cells in the side plates (field of view under 10X microscope).
FIG. 4 is a graph of the morphological characteristics of cells at day 4 during induction of the formation of mesodermal cells in the lateral plates (field of view under 10X microscope).
FIG. 5 is a graph of the cell morphology at day 6 in the induction of high endothelial cell propensity endothelial progenitor cells by the method of the present invention (10X microscopic field).
FIG. 6 is a graph of the morphological characteristics of cells at day 6 of the induction of endothelial progenitor cells by the prior art method (10X field of microscopy).
FIG. 7 is a graph of cellular immunolabeling at day 6 (10X microscopic field) during the induction of high endothelial cell propensity endothelial progenitor formation by the methods of the present invention.
FIG. 8 is a graph of cellular immunolabeling at day 6 (10X microscopic field) during endothelial progenitor cell-induced formation by a prior art method.
FIG. 9 is a graph of cell morphology at day 7 during the induction of mature endothelial cells (10X microscopic field).
FIG. 10 is a graph of cell morphology at day 9 during the induction of mature endothelial cells (10X field of microscopy).
FIG. 11 is an in vitro tube formation ability assay (10X microscopic field) of mature endothelial cells.
Detailed Description
The principles and features of this invention are described below in conjunction with specific embodiments, which are set forth merely to illustrate the invention and are not intended to limit the scope of the invention.
Example 1
The specific process of the method for efficiently differentiating induced pluripotent stem cells into mature endothelial cells of the embodiment is shown in fig. 1, and comprises the following steps:
step 1: and (3) pretreating the induced pluripotent stem cells to obtain a cell resuspension. The specific method comprises the following steps:
in a cell culture plate, after the induced pluripotent stem cells are recovered and passaged, the induced pluripotent stem cells are washed for 2 times by PBS (phosphate buffer solution), and then the concentration of the induced pluripotent stem cells is 10cm per minute2Adding 0.5mL of liquid into the bottom area of the cell culture plate, and adding a cell digestive juice preheated at 37 ℃ for enzymolysis digestion to obtain an enzymolysis digestive juice; when 70-80% of the induced pluripotent stem cells begin to shed, adding DMEM/F12 culture medium with 5 times volume of the enzymolysis digestion solution for neutralization, and then transferring the induced pluripotent stem cells into a centrifuge tube. Wherein the DMEM/F12 medium was purchased from Life Technologies, Inc. model number 11039021.
Centrifuging at room temperature for 5min at 200g, removing supernatant, adding TeSR-E8 culture medium, and resuspending cell pellet to obtain cell resuspension solution.
Wherein, the method for recovering and passaging the induced pluripotent stem cells comprises the following steps:
the cell culture plate coated with basement membrane matrix was prepared one day before recovery and placed at 37 ℃ in 5% v/v CO2And (4) in a cell culture box, performing overnight incubation to obtain a cell culture plate coated with the basement membrane substrate after overnight incubation.
The induced pluripotent stem cells are obtained by inducing somatic cells by using an induction kit, wherein the induction kit is Epi5TMEpisomalt iPSC replication Kit, available from Saimer Feishale science, MAN 0008352.
Removing cryopreserved induced pluripotent stem cells from liquid nitrogen, inoculating the cells in the overnight incubated basement membrane substrate-coated cell culture plate, and placing the cells in a 5% v/v CO solution at 37 deg.C2And (4) incubating for 16-24 hours in a cell incubator.
And then replacing a fresh TeSR-E8 culture medium preheated at 37 ℃, and repeatedly changing the culture medium every day until the cell polymerization degree is increased to 80 percent, thereby obtaining the recovered induced pluripotent stem cells.
And (3) carrying out passage on the recovered induced pluripotent stem cells, replacing a fresh TeSR-E8 culture medium preheated at 37 ℃ after one day, and repeatedly replacing the culture medium every day until the cell polymerization degree is increased to 80%, thus obtaining the induced pluripotent stem cells after passage.
Step 2: the cell resuspension obtained in step 1 is added according to the proportion of each 1cm2The cell culture plate of (2) was seeded with 3.5 ten thousand cells in the bottom area, transferred to a cell culture plate containing a mixture of TeSR-E8 medium and ROCK inhibitor Y27632, and placed at 37 ℃ with 5% v/v CO2The cells were cultured in a cell incubator for 20 hours. Wherein, in the mixture of the TeSR-E8 culture medium and the ROCK inhibitor Y27632, the concentration of the ROCK inhibitor Y27632 is 10 mu M/L. Cell morphology at induction day 1 is shown in figure 2.
And step 3: removing the old medium from the cell culture plate in step 2 at a rate of every 10cm2Cell culture ofAdding 4mL of the first N2B27 insulin deletion medium into the culture plate at 37 deg.C and 5% v/v CO2The cells were cultured in a cell incubator for 20 hours. The first N2B27 insulin deficiency culture medium is formed by adding BMP4, CHIR-99021 and Activin A into an N2B27 insulin deficiency culture medium, wherein the concentration of BMP4 is 25ng/mL, the concentration of CHIR-99021 is 8 mu M/L, and the concentration of Activin is 50 ng/mL. Cell morphology at induction day 2 is shown in figure 3.
And 4, step 4: removing the old medium from the cell culture plate in step 3, and washing the cell culture plate with PBS every 10cm2The amount of the second N2B27 insulin deletion medium added to the cell culture plate was 6mL at 37 ℃ in 5% v/v CO2After culturing in the cell culture box for 48 hours, the mesoderm cells of the side plate were obtained in the cell culture plate. Wherein the second N2B27 insulin deficiency medium is N2B27 insulin deficiency medium added with BMP4 and CHIR-99021, wherein the concentration of BMP4 is 25ng/mL, and the concentration of CHIR-99021 is 8 mu M/L. Cell morphology at induction day 4 is shown in figure 4.
And 5: the old medium in the cell culture plate containing the side plate mesoderm cells in step 4 was removed, and the cell culture plate was washed with PBS at every 10cm2The amount of the solution added to the bottom area of the cell culture plate was 4mL, and the first StemPro-34 medium was added thereto, followed by incubation at 37 ℃ and 5% v/v CO2After culturing in the cell culture box for 20 hours, repeating the step 5 once, and inducing endothelial progenitor cells with endothelial cell tendency in the cell culture plate. Wherein, the first StemPro-34 culture medium is obtained by adding VEGFA, Forskolin and SB431542 into the StemPro-34 culture medium, wherein the concentration of the VEGFA is 200ng/mL, the concentration of the Forskolin is 2 muM/L, and the concentration of the SB431542 is 10 muM/L.
Step 6: removing the old medium from the cell culture plate containing endothelial progenitor cells that are prone to endothelial cell in step 5, and washing the cell culture plate with PBS at a rate of every 10cm2Adding 0.5mL of cell culture plate bottom area solution, adding cell decomposition enzyme mixed solution composed of cell digestive juice and trypsin at volume ratio of 1:1, performing enzymolysis to obtain cell culture plate containing endothelial cellsEndothelial progenitor cells. Day 6 of induction, i.e., induction of endothelial progenitor cells with a higher endothelial cell propensity than the original method, the state under the cytoscope is shown in fig. 5, and no longer has a significant "ring hill" structure (as shown in fig. 6) compared with the original method; it can be seen by cellular immunolabeling (as shown in FIGS. 7 and 8) that the method of the invention yielded mixed cells with a smaller proportion of late endothelial progenitor cells (indicated by black arrows) and more proximal mature endothelial cells (indicated by white arrows) on day 6.
And 7: the endothelial progenitor cells which are obtained in the step 6 and have the endothelial cell tendency after enzymolysis are distributed according to the proportion of every 10cm2The amount of the solution added to the bottom area of the cell culture plate is 1mL, and a second StemPro-34 culture medium is added to obtain the endothelial progenitor cell resuspension containing endothelial cell tendency. Wherein, the second StemPro-34 culture medium is added with VEGFA in the StemPro-34 culture medium, wherein, the concentration of VEGFA is 50 ng/mL.
And 8: re-suspending the endothelial progenitor cell resuspension containing the endothelial cell inclination obtained in the step 7 according to the proportion of each 1cm2The cell culture plate of (2) was seeded with 4.5 ten thousand cells in the bottom area, transferred to a cell differentiation culture plate containing a second StemPro-34 medium, and placed at 37 ℃ with 5% v/v CO2The cells were cultured in a cell incubator for 20 hours. Wherein the cell differentiation culture plate containing the second StemPro-34 medium is obtained by the following method: per 10cm2The amount of the solution added to the bottom area of the cell culture plate is 2mL, 10 mu g/mL of fibronectin working solution is added into the cell culture plate, the cell culture plate is kept stand at room temperature for coating for 1 hour, the fibronectin working solution is removed, and the concentration of the fibronectin working solution per 10cm is determined2The amount of the added liquid on the bottom area of the cell culture plate is 2mL, and a second StemPro-34 culture medium is added, thus obtaining the cell differentiation culture plate containing the second StemPro-34 culture medium. After 20 hours, the state under the lens is shown in FIG. 9.
And step 9: removing the old medium from the cell culture plate in step 8, and washing the cell culture plate with PBS at a rate of every 10cm2The amount of the solution added to the bottom area of the cell culture plate was 2mL, and a second StemPro-34 medium was added thereto, and the mixture was incubated at 37 ℃ and 5% v/v CO2Cell cultureCulturing for 48 hours in a box to obtain mature induced differentiation endothelial cells. The state under the cell mirror is shown in FIG. 10. The resulting mature endothelial cells had significant in vitro tube forming ability as shown in figure 11.
Therefore, the invention can efficiently induce pluripotent stem cells to directionally differentiate into endothelial cells by increasing, decreasing and matching various cell induction factors and reasonably upgrading the induction method, and can obtain a large amount of mature endothelial cells with high purity and complete functions under the conditions of adopting a small amount of induced pluripotent stem cells and not needing purification operation. Meanwhile, the whole induction process is short in time consumption and simple to operate, animal-derived cells and components are not involved, the high efficiency, the functionality and the safety of the endothelial cells derived from the induced pluripotent stem cells are greatly improved, and a new way and a new method are provided for producing mature endothelial cells at the clinical application level on a large scale by utilizing the induced pluripotent stem cells of a patient.
Example 2
The specific process of the method for efficiently differentiating induced pluripotent stem cells into mature endothelial cells of the embodiment is shown in fig. 1, and comprises the following steps:
step 1: and (3) pretreating the induced pluripotent stem cells to obtain a cell resuspension. The specific method comprises the following steps:
in a cell culture plate, after the induced pluripotent stem cells are recovered and passaged, the induced pluripotent stem cells are washed for 2 times by PBS (phosphate buffer solution), and then the concentration of the induced pluripotent stem cells is 10cm per minute2Adding 0.5mL of liquid into the bottom area of the cell culture plate, and adding a cell digestive juice preheated at 37 ℃ for enzymolysis digestion to obtain an enzymolysis digestive juice; when 70% of the induced pluripotent stem cells begin to shed, adding DMEM/F12 culture medium with 5 times volume of the enzymolysis digestion solution for neutralization, and then transferring the induced pluripotent stem cells into a centrifuge tube. The DMEM/F12 medium was purchased from Life Technologies, Inc. and was designated 11039021.
Centrifuging at room temperature for 5min at 200g, removing supernatant, adding TeSR-E8 culture medium, and resuspending cell pellet to obtain cell resuspension solution.
Wherein, the method for recovering and passaging the induced pluripotent stem cells comprises the following steps:
the cell culture plate coated with basement membrane matrix was prepared one day before recovery and placed at 37 ℃ in 5% v/v CO2And (4) in a cell culture box, performing overnight incubation to obtain a cell culture plate coated with the basement membrane substrate after overnight incubation.
The induced pluripotent stem cells are obtained by inducing somatic cells by using an induction kit, wherein the induction kit is Epi5TMEpisomalt iPSC replication Kit, available from Saimer Feishale science, MAN 0008352.
Removing cryopreserved induced pluripotent stem cells from liquid nitrogen, inoculating the cells in the overnight incubated basement membrane substrate-coated cell culture plate, and placing the cells in a 5% v/v CO solution at 37 deg.C2In the cell incubator, incubation was performed for 16 hours.
And then replacing a fresh TeSR-E8 culture medium preheated at 37 ℃, and repeatedly changing the culture medium every day until the cell polymerization degree is increased to 75 percent, thereby obtaining the recovered induced pluripotent stem cells.
And (3) carrying out passage on the recovered induced pluripotent stem cells, replacing a fresh TeSR-E8 culture medium preheated at 37 ℃ after one day, and repeatedly replacing the culture medium every day until the cell polymerization degree is increased to 75%, thus obtaining the induced pluripotent stem cells after passage.
Step 2: the cell resuspension obtained in step 1 is added according to the proportion of each 1cm2The cell culture plate of (2) ten thousand cells were plated on the bottom surface, transferred to a cell culture plate containing a mixture of TeSR-E8 medium and ROCK inhibitor Y27632, and placed at 37 ℃ with 5% v/v CO2The cells were cultured in a cell incubator for 24 hours. Wherein, in the mixture of the TeSR-E8 culture medium and the ROCK inhibitor Y27632, the concentration of the ROCK inhibitor Y27632 is 10 mu M/L.
And step 3: removing the old medium from the cell culture plate in step 2 at a rate of every 10cm2The cell culture plate (2) was added with a first N2B27 insulin deletion medium at a rate of 4mL in terms of the amount of liquid added to the bottom area of the plate, and the plate was incubated at 37 ℃ with 5% v/v CO2The cells were cultured in a cell incubator for 24 hours. The first N2B27 insulin deficiency culture medium is formed by adding BMP4, CHIR-99021 and Activin A into an N2B27 insulin deficiency culture medium, wherein the concentration of BMP4 is 20ng/mL, the concentration of CHIR-99021 is 6 mu M/L, and the concentration of Activin is concentratedThe degree is 30 ng/mL.
And 4, step 4: removing the old medium from the cell culture plate in step 3, and washing the cell culture plate with PBS every 10cm2The amount of the second N2B27 insulin deletion medium added to the cell culture plate was 6mL at 37 ℃ in 5% v/v CO2After culturing in the cell culture box for 48 hours, the mesoderm cells of the side plate were obtained in the cell culture plate. Wherein the second N2B27 insulin deficiency medium is N2B27 insulin deficiency medium added with BMP4 and CHIR-99021, wherein the concentration of BMP4 is 20ng/mL, and the concentration of CHIR-99021 is 6 mu M/L.
And 5: the old medium in the cell culture plate containing the side plate mesoderm cells in step 4 was removed, and the cell culture plate was washed with PBS at every 10cm2The amount of the solution added to the bottom area of the cell culture plate was 4mL, and the first StemPro-34 medium was added thereto, followed by incubation at 37 ℃ and 5% v/v CO2After culturing for 24 hours in the cell culture box, repeating the step 5 once, and inducing endothelial progenitor cells with endothelial cell tendency in the cell culture plate. Wherein, the first StemPro-34 culture medium is obtained by adding VEGFA, Forskolin and SB431542 into the StemPro-34 culture medium, wherein the concentration of the VEGFA is 100ng/mL, the concentration of the Forskolin is 1 muM/L, and the concentration of the SB431542 is 5 muM/L.
Step 6: removing the old medium from the cell culture plate containing endothelial progenitor cells that are prone to endothelial cell in step 5, and washing the cell culture plate with PBS at a rate of every 10cm2The liquid adding amount of the cell culture plate bottom area is 0.5mL, and a cell decomposition enzyme mixed solution consisting of cell digestive juice and trypsin according to the volume ratio of 1:1 is added for enzymolysis, so that the endothelial progenitor cells with endothelial cell tendency after enzymolysis are obtained.
And 7: the endothelial progenitor cells which are obtained in the step 6 and have the endothelial cell tendency after enzymolysis are distributed according to the proportion of every 10cm2The amount of the solution added to the bottom area of the cell culture plate is 1mL, and a second StemPro-34 culture medium is added to obtain the endothelial progenitor cell resuspension containing endothelial cell tendency. Wherein, the second StemPro-34 culture medium is added with VEGFA in the StemPro-34 culture medium, wherein, the concentration of the VEGFA is 30 ng/mL.
And 8: re-suspending the endothelial progenitor cell resuspension containing the endothelial cell inclination obtained in the step 7 according to the proportion of each 1cm2The cell culture plate (2) was seeded with 3 ten thousand cells in the bottom area, transferred to a cell differentiation culture plate containing a second StemPro-34 medium, and placed at 37 ℃ with 5% v/v CO2Culturing in a cell culture box for 16-24 hours. Wherein the cell differentiation culture plate containing the second StemPro-34 medium is obtained by the following method: per 10cm2The amount of the solution added to the bottom area of the cell culture plate is 2mL, 10 mu g/mL of fibronectin working solution is added into the cell culture plate, the cell culture plate is kept stand at room temperature for coating for 1 hour, the fibronectin working solution is removed, and the concentration of the fibronectin working solution per 10cm is determined2The amount of the added liquid on the bottom area of the cell culture plate is 2mL, and a second StemPro-34 culture medium is added, thus obtaining the cell differentiation culture plate containing the second StemPro-34 culture medium.
And step 9: removing the old medium from the cell culture plate in step 8, and washing the cell culture plate with PBS at a rate of every 10cm2The amount of the solution added to the bottom area of the cell culture plate was 2mL, and a second StemPro-34 medium was added thereto, and the mixture was incubated at 37 ℃ and 5% v/v CO2Culturing for 48 hours in a cell culture box to obtain mature induced differentiation endothelial cells. The obtained mature endothelial cells have obvious in-vitro tube forming capacity.
Example 3
The specific process of the method for efficiently differentiating induced pluripotent stem cells into mature endothelial cells of the embodiment is shown in fig. 1, and comprises the following steps:
step 1: and (3) pretreating the induced pluripotent stem cells to obtain a cell resuspension. The specific method comprises the following steps:
in a cell culture plate, after the induced pluripotent stem cells are recovered and passaged, the induced pluripotent stem cells are washed for 2 times by PBS (phosphate buffer solution), and then the concentration of the induced pluripotent stem cells is 10cm per minute2Adding 0.5mL of liquid into the bottom area of the cell culture plate, and adding a cell digestive juice preheated at 37 ℃ for enzymolysis digestion to obtain an enzymolysis digestive juice; when 80% of induced pluripotent stem cells begin to shed, adding DMEM/F12 culture medium with 5 times volume of the enzymolysis digestive solution for neutralization, and then adding the induced pluripotent stem cellsThe pluripotent stem cells were transferred to a centrifuge tube. The DMEM/F12 medium was purchased from Life Technologies, Inc. and was designated 11039021.
Centrifuging at room temperature for 5min at 200g, removing supernatant, adding TeSR-E8 culture medium, and resuspending cell pellet to obtain cell resuspension solution.
Wherein, the method for recovering and passaging the induced pluripotent stem cells comprises the following steps:
the cell culture plate coated with basement membrane matrix was prepared one day before recovery and placed at 37 ℃ in 5% v/v CO2And (4) in a cell culture box, performing overnight incubation to obtain a cell culture plate coated with the basement membrane substrate after overnight incubation.
The induced pluripotent stem cells are obtained by inducing somatic cells by using an induction kit, wherein the induction kit is Epi5TMEpisomalt iPSC replication Kit, available from Saimer Feishale science, MAN 0008352.
Removing cryopreserved induced pluripotent stem cells from liquid nitrogen, inoculating the cells in the overnight incubated basement membrane substrate-coated cell culture plate, and placing the cells in a 5% v/v CO solution at 37 deg.C2In the cell incubator, incubation was performed for 24 hours.
And then replacing a fresh TeSR-E8 culture medium preheated at 37 ℃, and repeatedly changing the culture medium every day until the cell polymerization degree is increased to 85 percent, thereby obtaining the recovered induced pluripotent stem cells.
And (3) carrying out passage on the recovered induced pluripotent stem cells, replacing a fresh TeSR-E8 culture medium preheated at 37 ℃ after one day, and repeatedly replacing the culture medium every day until the cell polymerization degree is increased to 85%, thus obtaining the induced pluripotent stem cells after passage.
Step 2: the cell resuspension obtained in step 1 is added according to the proportion of each 1cm2The cell culture plate (2) was plated at a ratio of 5 ten thousand cells on the bottom surface, transferred to a cell culture plate containing a mixture of TeSR-E8 medium and ROCK inhibitor Y27632, and placed at 37 ℃ with 5% v/v CO2The cells were cultured in a cell incubator for 16 hours. Wherein, in the mixture of the TeSR-E8 culture medium and the ROCK inhibitor Y27632, the concentration of the ROCK inhibitor Y27632 is 10 mu M/L.
And step 3: removing the old medium from the cell culture plate in step 2,per 10cm2The cell culture plate (2) was added with a first N2B27 insulin deletion medium at a rate of 4mL in terms of the amount of liquid added to the bottom area of the plate, and the plate was incubated at 37 ℃ with 5% v/v CO2The cells were cultured in a cell incubator for 16 hours. The first N2B27 insulin deficiency medium is formed by adding BMP4, CHIR-99021 and Activin A into an N2B27 insulin deficiency medium, wherein the concentration of BMP4 is 30ng/mL, the concentration of CHIR-99021 is 10 mu M/L, and the concentration of Activin A is 70 ng/mL.
And 4, step 4: removing the old medium from the cell culture plate in step 3, and washing the cell culture plate with PBS every 10cm2The amount of the second N2B27 insulin deletion medium added to the cell culture plate was 6mL at 37 ℃ in 5% v/v CO2After culturing in the cell culture box for 48 hours, the mesoderm cells of the side plate were obtained in the cell culture plate. Wherein the second N2B27 insulin deficiency medium is N2B27 insulin deficiency medium added with BMP4 and CHIR-99021, wherein the concentration of BMP4 is 30ng/mL, and the concentration of CHIR-99021 is 10 mu M/L.
And 5: the old medium in the cell culture plate containing the side plate mesoderm cells in step 4 was removed, and the cell culture plate was washed with PBS at every 10cm2The amount of the solution added to the bottom area of the cell culture plate was 4mL, and the first StemPro-34 medium was added thereto, followed by incubation at 37 ℃ and 5% v/v CO2Culturing in a cell culture box for 16 hours, repeating the step 5 once, and inducing endothelial progenitor cells with endothelial cell tendency in a cell culture plate. Wherein, the first StemPro-34 culture medium is obtained by adding VEGFA, Forskolin and SB431542 into the StemPro-34 culture medium, wherein the concentration of the VEGFA is 300ng/mL, the concentration of the Forskolin is 4 mu M/L, and the concentration of the SB431542 is 15 mu M/L.
Step 6: removing the old medium from the cell culture plate containing endothelial progenitor cells that are prone to endothelial cell in step 5, and washing the cell culture plate with PBS at a rate of every 10cm2The liquid adding amount of the cell culture plate bottom area is 0.5mL, and a cell decomposition enzyme mixed solution consisting of cell digestive juice and trypsin according to the volume ratio of 1:1 is added for enzymolysis, so that the endothelial progenitor cells with endothelial cell tendency after enzymolysis are obtained.
And 7: the endothelial progenitor cells which are obtained in the step 6 and have the endothelial cell tendency after enzymolysis are distributed according to the proportion of every 10cm2The amount of the solution added to the bottom area of the cell culture plate is 1mL, and a second StemPro-34 culture medium is added to obtain the endothelial progenitor cell resuspension containing endothelial cell tendency. Wherein, the second StemPro-34 culture medium is added with VEGFA in the StemPro-34 culture medium, wherein, the concentration of the VEGFA is 70 ng/mL.
And 8: re-suspending the endothelial progenitor cell resuspension containing the endothelial cell inclination obtained in the step 7 according to the proportion of each 1cm2The cell culture plate (2) was seeded with 6 ten thousand cells in the bottom area, transferred to a cell differentiation culture plate containing a second StemPro-34 medium, and placed at 37 ℃ with 5% v/v CO2The cells were cultured in a cell incubator for 16 hours. Wherein the cell differentiation culture plate containing the second StemPro-34 medium is obtained by the following method: per 10cm2The amount of the solution added to the bottom area of the cell culture plate is 2mL, 10 mu g/mL of fibronectin working solution is added into the cell culture plate, the cell culture plate is kept stand at room temperature for coating for 1 hour, the fibronectin working solution is removed, and the concentration of the fibronectin working solution per 10cm is determined2The amount of the added liquid on the bottom area of the cell culture plate is 2mL, and a second StemPro-34 culture medium is added, thus obtaining the cell differentiation culture plate containing the second StemPro-34 culture medium.
And step 9: removing the old medium from the cell culture plate in step 8, and washing the cell culture plate with PBS at a rate of every 10cm2The amount of the solution added to the bottom area of the cell culture plate was 2mL, and a second StemPro-34 medium was added thereto, and the mixture was incubated at 37 ℃ and 5% v/v CO2Culturing for 48 hours in a cell culture box to obtain mature induced differentiation endothelial cells. The obtained mature endothelial cells have obvious in-vitro tube forming capacity.
Step 10: and (4) expanding and freezing the obtained mature induced differentiation endothelial cells.
The specific method for amplifying the obtained mature induced differentiation endothelial cells comprises the following steps:
when the cell polymerization degree is expanded to 80-90%, the passage operation can be started.
Taking out cell cultureThe plates were incubated, the old medium was aspirated, washed 2 times with PBS at room temperature, and then every 10cm2The amount of the added liquid in the bottom area of the cell culture plate is 0.5mL, a cell-decomposing enzyme mixed solution which is preheated at 37 ℃ and consists of cell digestive juice and trypsin according to the volume ratio of 1:1 is added, and the cell-decomposing enzyme mixed solution is placed at 37 ℃ and 5% v/v CO2The cell culture box is maintained for 2-3min until most of the cells begin to float and fall off in the form of lumps.
Per 10cm2The liquid adding amount of the cell culture plate bottom area is 1mL, the first StemPro-34 culture medium preheated at 37 ℃ is added to neutralize the enzymolysis of cell digestive juice and trypsin, the exfoliated cell mass is lightly blown by a pipette gun to be resuspended, and then the cell culture plate is transferred to a 15mL centrifuge tube.
At room temperature, 200g, centrifuge for 5 min. The supernatant was removed with a pipette gun, followed by 3mL of a second StemPro-34 medium pre-warmed at 37 ℃ and gently pipetting to resuspend the cell pellet well.
Per 10cm2The amount of the solution added to the bottom area of the cell culture plate was 2mL, and a second StemPro-34 medium preheated to 37 ℃ was added to the cell culture plate. Subsequently, 3mL of the cell resuspension was aliquoted into cell culture plates at 1:3 or 1:2 passages, placed at 37 ℃ in 5% v/v CO2And (4) performing static culture on the cell culture box.
The specific method for cryopreserving the obtained mature induced differentiation endothelial cells comprises the following steps:
observing the cells under a mirror, determining that the EC cells have no pollution and good state, and performing cryopreservation operation on the EC cells when the polymerization degree reaches 80-90%.
The cell culture plate was removed, the old medium was aspirated, washed 2 times with PBS at room temperature, and then washed every 10cm2The amount of the added liquid in the bottom area of the cell culture plate is 0.5mL, a cell-decomposing enzyme mixed solution which is preheated at 37 ℃ and consists of cell digestive juice and trypsin according to the volume ratio of 1:1 is added, and the cell-decomposing enzyme mixed solution is placed at 37 ℃ and 5% v/v CO2The cell culture box is maintained for 2-3min until most of the cells begin to float and fall off in the form of lumps.
Per 10cm2The ratio of the amount of liquid added to the bottom area of the cell culture plate of 1mLA second StemPro-34 medium, pre-warmed at 37 deg.C, was added to neutralize the enzymatic action of the cell digest and trypsin, and the detached cell pellet was gently pipetted into a 15mL centrifuge tube for resuspension and subsequent transfer.
Centrifuge at 200g for 5min at room temperature. During the period, a cryopreservation tube is prepared and marked (name of culture person, cryopreservation time, culture generation number, etc.).
The supernatant was aspirated off with a pipette gun, followed by addition of 2mL of the cryo-SFM stock solution, gentle pipetting to thoroughly resuspend the cell pellet, and then aliquoting into cryotubes at 1mL per tube (a 1:2 ratio is recommended for cryopreservation).
And (3) putting the freezing tube into a program cooling box with pre-cooling at 4 ℃, putting the box into a refrigerator with the temperature of 80 ℃ below zero, and transferring the box into a liquid nitrogen tank for storage the next day.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (6)
1. A method for efficiently differentiating induced pluripotent stem cells into mature endothelial cells is characterized by comprising the following steps:
step 1: pretreating induced pluripotent stem cells to obtain cell resuspension;
step 2: the cell resuspension obtained in step 1 is added according to the proportion of each 1cm2The bottom area of the cell culture plate is paved with 2-5 ten thousand cells, the cells are transferred into a cell culture plate containing a mixture of TeSR-E8 culture medium and ROCK inhibitor Y27632, and the cell culture plate is placed in a cell culture box for culturing for 16-24 hours;
and step 3: removing the old medium from the cell culture plate in step 2 at a rate of every 10cm2The liquid adding amount of the bottom area of the cell culture plate is 4mL, a first N2B27 insulin deficiency culture medium is added, and the cell culture plate is placed in a cell culture box for culture for 16 to 24 hours; the first N2B27 insulin deficiency culture medium is formed by adding BMP4, CHIR-99021 and Activin A into an N2B27 insulin deficiency culture medium, wherein the concentration of BMP4 is 20-30ng/mL, and the concentration of CHIR-99021 is 6-10 mu M ^ HL, the concentration of Activin A is 30-70 ng/mL;
and 4, step 4: removing the old medium from the cell culture plate in step 3, and washing the cell culture plate with PBS every 10cm2Adding a second N2B27 insulin deletion culture medium into the cell culture plate at a ratio of 6mL of liquid adding amount of the bottom area of the cell culture plate, placing the cell culture plate in a cell culture box for culturing for 48 hours, and obtaining lateral plate mesoderm cells in the cell culture plate; the second N2B27 insulin deletion medium is formed by adding BMP4 and CHIR-99021 into N2B27 insulin deletion medium, wherein the concentration of BMP4 is 20-30ng/mL, and the concentration of CHIR-99021 is 6-10 mu M/L;
and 5: the old medium in the cell culture plate containing the side plate mesoderm cells in step 4 was removed, and the cell culture plate was washed with PBS at every 10cm2Adding a first StemPro-34 culture medium into the cell culture plate with the liquid adding amount of 4mL, then placing the cell culture plate into a cell culture box for culturing for 16-24 hours, repeating the operation of the step 5 once, and inducing in the cell culture plate to obtain endothelial progenitor cells with endothelial cell tendency; the first StemPro-34 culture medium is obtained by adding VEGFA, Forskolin and SB431542 into the StemPro-34 culture medium, wherein the concentration of VEGFA is 100-300ng/mL, the concentration of Forskolin is 1-4 muM/L, and the concentration of SB431542 is 5-15 muM/L;
step 6: removing the old medium from the cell culture plate containing endothelial progenitor cells that are prone to endothelial cell in step 5, and washing the cell culture plate with PBS at a rate of every 10cm2The liquid adding amount of the cell culture plate bottom area is 0.5mL, and cell-decomposing enzyme mixed liquor is added for enzymolysis to obtain endothelial progenitor cells which are subjected to enzymolysis and have endothelial cell tendency;
and 7: the endothelial progenitor cells which are obtained in the step 6 and have the endothelial cell tendency after enzymolysis are distributed according to the proportion of every 10cm2Adding a second StemPro-34 culture medium into the cell culture plate with the liquid adding amount of 1mL to obtain endothelial progenitor cell resuspension containing endothelial cell tendency; the second StemPro-34 culture medium is obtained by adding VEGFA into the StemPro-34 culture medium, wherein the concentration of VEGFA is 30-70 ng/mL;
and 8: the endothelial progenitor cells which are obtained in the step 7 and have the tendency of endothelial cellsResuspending in 1cm of suspension2The bottom area of the cell culture plate is paved with 3-6 ten thousand cells, the cells are transferred into a cell differentiation culture plate containing a second StemPro-34 culture medium, and the cells are placed in a cell culture box for culture for 16-24 hours;
and step 9: removing the old medium from the cell culture plate in step 8, and washing the cell culture plate with PBS at a rate of every 10cm2The liquid adding amount of the bottom area of the cell culture plate is 2mL, a second StemPro-34 culture medium is added, and the cell culture plate is placed in a cell culture box and cultured for 48 hours to obtain mature induced differentiation endothelial cells.
2. The method of claim 1, wherein the step 1 of pretreating the induced pluripotent stem cells to obtain the cell resuspension comprises:
in a cell culture plate, after the induced pluripotent stem cells are recovered and passaged, the induced pluripotent stem cells are washed for 2 times by PBS (phosphate buffer solution), and then the concentration of the induced pluripotent stem cells is 10cm per minute2Adding 0.5mL of liquid into the bottom area of the cell culture plate, and adding a cell digestive juice preheated at 37 ℃ for enzymolysis digestion to obtain an enzymolysis digestive juice; when 70-80% of induced pluripotent stem cells begin to fall off, adding DMEM/F12 culture medium with the volume 5 times that of the enzymolysis digestion solution for neutralization, then transferring the induced pluripotent stem cells into a centrifuge tube, centrifuging, removing supernatant, adding TeSR-E8 culture medium, and carrying out heavy suspension on the cell sediment to obtain cell heavy suspension.
3. The method for inducing the efficient differentiation of pluripotent stem cells into mature endothelial cells according to claim 1, wherein in step 2, the concentration of ROCK inhibitor Y27632 in the mixture of TeSR-E8 medium and ROCK inhibitor Y27632 is 10 μ M/L.
4. The method of claim 1, wherein in step 6, the mixture of cell lytic enzymes comprises a cell digest and trypsin at a volume ratio of 1: 1.
5. The method of claim 1, wherein in step 8, the cell differentiation culture plate containing the second StemPro-34 medium is obtained by the following method: per 1cm2The amount of the solution added to the bottom area of the cell culture plate is 2mL, 10 mu g/mL of fibronectin working solution is added into the cell culture plate, the cell culture plate is kept stand at room temperature for coating for 1 hour, the fibronectin working solution is removed, and the concentration of the fibronectin working solution per 10cm is determined2The amount of the added liquid on the bottom area of the cell culture plate is 2mL, and a second StemPro-34 culture medium is added, thus obtaining the cell differentiation culture plate containing the second StemPro-34 culture medium.
6. The method for inducing the efficient differentiation of pluripotent stem cells into mature endothelial cells according to any one of claims 1 to 5, wherein the culture conditions of the cell culture chamber are 37 ℃ and 5% v/v CO2。
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