CN112126625A - In-vitro amplification method of limbal stem cells - Google Patents

In-vitro amplification method of limbal stem cells Download PDF

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CN112126625A
CN112126625A CN202011059056.6A CN202011059056A CN112126625A CN 112126625 A CN112126625 A CN 112126625A CN 202011059056 A CN202011059056 A CN 202011059056A CN 112126625 A CN112126625 A CN 112126625A
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limbal stem
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湛振键
齐国光
刘世豪
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Guangdong Zhikang Intelligent Biotechnology Co ltd
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Guangdong Kangdun Bioengineering Technology Co Ltd
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Abstract

The invention provides an in-vitro amplification method of limbal stem cells, which comprises the following steps: (1) shearing and washing LSCs stem cell tissue blocks, adding 90-95% of LSCs stem cell tissue by mass into 30-50 times of volume of digestive juice for digestion, washing, and adding 8-10 times of volume of LSCs stem cell culture medium; (2) adding a tissue protective agent with 0.1-0.5 times of volume, centrifuging, and removing supernatant; (3) washing with Hank solution, resuspending, and planting the tissue mixed with 5-10% undigested limbal stem cells into a porous culture plate for culture to obtain limbal stem cells; the invention can effectively protect the discrete limbal stem cells in the enzymolysis separation process, maintain the cell proliferation activity, maintain the stable proliferation activity of the cells in the separation process and realize the overall efficient and stable in-vitro amplification culture of the limbal stem cells.

Description

In-vitro amplification method of limbal stem cells
Technical Field
The invention relates to the technical field of cell culture, in particular to an in-vitro amplification method of limbal stem cells.
Background
With the continuous and deep research of biological tissue engineering, the technology of reconstructing corneal epithelium is important in the tissue engineering of cornea. Limbal Stem Cells (LSCs) are the interface of the cornea, conjunctiva and sclera, and healing of the corneal epithelium is accomplished by migration and proliferation of limbal stem cells. Therefore, in vitro culture of limbal stem cells is critical for the reconstruction of corneal epithelium.
However, the construction of the existing in vitro culture system for the limbal stem cells has more difficulties, and in the process of the in vitro amplification culture of the limbal stem cells, the stability of the in vitro amplification of the limbal stem cells is difficult to maintain due to the influence of the separation culture process, so that the problems of slow proliferation speed of the limbal cells, long culture period, few passage numbers, easy pollution and the like are caused, the in vitro amplification effect of the limbal stem cells with high efficiency and stability cannot be achieved, and the proliferation potential of the in vitro amplification culture of the limbal stem cells is fully exerted. Therefore, a more effective method for in vitro expansion of the limbal stem cells is found, which provides an important basis for constructing an in vitro culture system of the limbal stem cells.
Disclosure of Invention
In view of this, the present invention provides an in vitro method for expanding limbal stem cells.
The technical scheme of the invention is realized as follows:
the invention provides an in-vitro amplification method of limbal stem cells, which comprises the following steps:
(1) cutting the limbal stem cell tissue blocks into pieces, flushing the limbal stem cell tissue blocks for 2-3 times by using Hank liquid in an aseptic environment, adding 30-50 times of digestive juice by volume into 90-95% of the limbal stem cell tissue by mass, digesting, and removing the digestive juice; after 2-3 times of flushing by Hank liquid, adding a limbus stem cell culture medium with the volume of 8-10 times, and stopping trypsinase digestion;
(2) adding a tissue protective agent with the volume 0.1-0.5 times that of the mixture, centrifuging for 8-10 min at the speed of 1000-1100 r/min, removing supernatant, and taking precipitated cells; the tissue protective agent is prepared by adopting a limbal stem cell basic culture solution DMEM and adding 0.55-1.25 ug/ml echinacoside, 1.5-2.5 mg/ml beta-glucan and 2.2-3.8 mg/ml L-glutamine for mixing;
(3) washing the precipitated cells for 1-2 times by using Hank solution, adding the cells into a limbal stem cell culture medium with the volume of 3-5 times, re-suspending, mixing with 5-10% of undigested limbal stem cell tissues, planting the mixture into a porous culture plate, and culturing at 36-37 ℃ and 93-95% of humidity at 5-5.5% of CO2Cultured in an incubatorAnd replacing the liquid every other day to obtain the limbal stem cells.
Further, in the step (1), before the addition of the limbal stem cell culture medium to terminate the trypsin digestion, a limbal stem cell basic culture solution DMEM/F12(1:1) containing 0.5-0.8% of fetal bovine serum is added, the mixture is placed in a constant temperature shaking table at 37 ℃ to react for 3-5 min, and the cell culture medium is sucked out. Pretreatment of limbal stem cells was performed by using a limbal stem cell basal medium DMEM/F12(1:1) containing a trace amount of fetal bovine serum, which was advantageous for better maintenance of cell proliferation activity.
Further, the tissue protective agent contains 1.05ug/ml echinacoside, 2.0mg/ml beta-glucan and 3.0mg/ml L-glutamine. The echinacoside and the beta-glucan with certain content are combined, so that the function of discrete limbal stem cells in the enzymolysis separation process is protected, the cell proliferation activity of the limbal cells in the in vitro separation process is maintained, and the cell proliferation stability is improved.
Further, the limbal stem cell culture medium is prepared by mixing a limbal stem cell basic culture solution DMEM/F12(1:1) with 10-15% fetal bovine serum, 2.5-3 ml of 100 Xstreptomycin mixed solution, 0.3-0.5 mg/ml glutamine, 5-8 ug/ml insulin, 15-25 mg/ml transferrin, 0.2-0.5 ug/ml hydrocortisone, 10-16 ng/ml cholera toxin, 3-5 ng/ml platelet-derived factor, 1-3 ng/ml interleukin-6 and 10-15 ng/ml insulin-like growth factor.
Further, in step (1), the digestion solution is obtained by mixing 1.25g/L trypsin and 0.2g/L EDTA1: 1.
Further, in the step (1), the consumption time is 13-15 min.
Further, in step (3), the density of the seeded resuspended cells in the well plate is 1X 105Cells/cm2。
Further, in step (3), 4 volumes of the limbal stem cell culture medium were resuspended and mixed with 8% undigested limbal stem cell tissue and plated into well-plated plates.
Compared with the prior art, the invention has the beneficial effects that: the method mainly optimizes the separation culture process of the limbal stem cells, adopts the limbal stem cell basic culture solution DMEM containing a certain amount of echinacoside, beta-glucan and L-glutamine as a protective agent for cell centrifugal precipitation on the basis of ensuring the full digestion of the limbal stem cells, achieves the effect of effectively protecting the discrete limbal stem cells in the enzymolysis separation process, reduces the pollution of the limbal stem cells in the in vitro separation culture, is favorable for better keeping the cell proliferation activity in the in vitro separation process, and is favorable for keeping the stable proliferation activity of the cells in the separation process. Meanwhile, the limbal stem cells combined and dispersed by trace undigested tissue blocks are subjected to amplification culture under certain conditions, so that the growth and proliferation of the limbal stem cells are better promoted under the condition of effectively maintaining the undifferentiated state of the limbal stem cells, the film-forming growth of cells is facilitated, the proliferation capacity is improved, and the characteristics of the limbal stem cells are maintained; thereby successfully realizing the overall efficient and stable in-vitro amplification culture of the limbal stem cells and fully exerting the proliferation potential of the in-vitro amplification culture of the limbal stem cells.
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FIG. 1 is a line graph showing the results of primary and serial 3-passage cell expansion in the method for in vitro expansion of limbal stem cells according to the present invention.
Detailed Description
In order to better understand the technical content of the invention, specific examples are provided below to further illustrate the invention.
The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.
The materials, reagents and the like used in the examples of the present invention can be obtained commercially without specific description.
Example 1-a method for the in vitro expansion of limbal stem cells comprising the steps of:
(1) cutting the limbal stem cell tissue block, flushing with Hank solution for 2 times in a sterile environment, adding 90% of limbal stem cell tissue into 30 times of digestive juice, digesting for 13min, and discarding the digestive juice; after 2 times of flushing by Hank liquid, in order to eliminate the digestion effect of EDTA, adding a limbal stem cell culture medium with 8 times of volume, and stopping trypsinase digestion;
wherein the digestive juice is prepared by mixing 1.25g/L trypsin and 0.2g/L EDTA1: 1; the limbal stem cell culture medium is prepared by mixing a limbal stem cell basic culture solution DMEM/F12(1:1), 10% fetal bovine serum, 2.5ml of 100 Xstreptomycin mixed solution, 0.3mg/ml glutamine, 5ug/ml insulin, 15mg/ml transferrin, 0.2ug/ml hydrocortisone, 10ng/ml cholera toxin, 3ng/ml platelet-derived factor, 1ng/ml interleukin-6 and 10ng/ml insulin-like growth factor;
(2) adding tissue protective agent 0.1 times volume, centrifuging at 1000r/min for 8min, removing supernatant, and collecting precipitated cells; the tissue protective agent is prepared by adopting a limbal stem cell basic culture solution DMEM, adding 0.55ug/ml echinacoside, 1.5mg/ml beta-glucan and 2.2mg/ml L-glutamine and mixing;
(3) washing the precipitated cells with Hank solution for 1 time, adding into 3 times of limbal stem cell culture medium, resuspending, and inoculating at density of 1 × 105Cells/cm2Mixed with 10% undigested limbal stem cell tissue, seeded into 10% matrigel-coated 6-well plates at 36 ℃ and 93% humidity with 5% CO2Culturing in an incubator, and changing the culture solution every other day to obtain the limbal stem cells.
Example 2-a method for the in vitro expansion of limbal stem cells comprising the steps of:
(1) cutting the limbal stem cell tissue block, flushing with Hank liquid in a sterile environment for 3 times, adding 95% of limbal stem cell tissue into 50 times of digestive juice, digesting for 15min, and discarding the digestive juice; washing with Hank solution for 3 times, adding 0.5% fetal calf serum-containing limbal stem cell basic culture solution DMEM/F12(1:1) to eliminate EDTA digestion, reacting in a constant temperature shaking table at 37 deg.C for 3min, and sucking out cell culture medium; then adding 10 times of the limbal stem cell culture medium, and stopping trypsinization;
wherein the digestive juice is prepared by mixing 1.25g/L trypsin and 0.2g/L EDTA1: 1; the limbal stem cell culture medium is prepared by mixing a limbal stem cell basic culture solution DMEM/F12(1:1), 15% fetal bovine serum, 3ml of 100 Xstreptomycin mixed solution, 0.5mg/ml glutamine, 8ug/ml insulin, 25mg/ml transferrin, 0.5ug/ml hydrocortisone, 16ng/ml cholera toxin, 5ng/ml platelet-derived factor, 3ng/ml interleukin-6 and 15ng/ml insulin-like growth factor;
(2) adding tissue protective agent 0.5 times volume, centrifuging at 1100r/min for 10min, removing supernatant, and collecting precipitated cells; the tissue protective agent is prepared by adopting a limbal stem cell basic culture solution DMEM, adding 1.25ug/ml echinacoside, 2.5mg/ml beta-glucan and 3.8mg/ml L-glutamine and mixing;
(3) washing the precipitated cells with Hank solution for 2 times, adding the cells into a 5-fold volume of limbal stem cell culture medium for resuspension, and inoculating the cells at a density of 1 × 105Cells/cm2Mixed with 5% undigested limbal stem cell tissue, seeded into 10% matrigel-coated 6-well plates at 37 ℃ and 95% humidity with 5.5% CO2Culturing in an incubator, and changing the culture solution every other day to obtain the limbal stem cells.
Example 3-a method for the in vitro expansion of limbal stem cells comprising the steps of:
(1) cutting the limbal stem cell tissue block, flushing with Hank solution for 3 times in a sterile environment, adding 92% of limbal stem cell tissue by mass into 40 times of digestive juice for digestion for 14min, and discarding the digestive juice; washing with Hank solution for 3 times, adding 0.8% fetal calf serum-containing limbal stem cell basic culture solution DMEM/F12(1:1) to eliminate EDTA digestion, reacting in a constant temperature shaking table at 37 deg.C for 5min, and sucking out cell culture medium; then adding 10 times of the limbal stem cell culture medium, and stopping trypsinization;
wherein the digestive juice is prepared by mixing 1.25g/L trypsin and 0.2g/L EDTA1: 1; the limbal stem cell culture medium is prepared by adopting a limbal stem cell basic culture solution DMEM/F12(1:1), adding 13% fetal bovine serum, 2.8ml of 100 Xstreptomycin mixed solution, 0.4mg/ml glutamine, 6ug/ml insulin, 20mg/ml transferrin, 0.3ug/ml hydrocortisone, 13ng/ml cholera toxin, 4ng/ml platelet-derived factor, 2ng/ml interleukin-6 and 12ng/ml insulin-like growth factor and mixing;
(2) adding tissue protective agent 0.3 times volume, centrifuging at 1100r/min for 10min, removing supernatant, and collecting precipitated cells; the tissue protective agent is prepared by adopting a limbal stem cell basic culture solution DMEM, adding 1.05ug/ml echinacoside, 2.0mg/ml beta-glucan and 3.0mg/ml L-glutamine and mixing;
(3) washing the precipitated cells with Hank solution for 2 times, adding into 4 times of limbal stem cell culture medium, re-suspending, and inoculating at a density of 1 × 105Cells/cm2Mixed with 8% undigested limbal stem cell tissue, seeded into 10% matrigel-coated 6-well plates at 37 ℃ and 95% humidity with 5% CO2Culturing in an incubator, and changing the culture solution every other day to obtain the limbal stem cells.
Example 4-a method for the in vitro expansion of limbal stem cells according to example 3, with the exception that: in the step (2), the tissue protective agent contains 1.0ug/ml echinacoside, 2.0mg/ml beta-glucan and 3.0mg/ml L-glutamine.
Example 5-a method for the in vitro expansion of limbal stem cells according to example 3, with the exception that: in the step (2), the tissue protective agent contains 1.15ug/ml echinacoside, 2.0mg/ml beta-glucan and 3.0mg/ml L-glutamine.
Comparative example 1-a method of in vitro expansion of limbal stem cells according to example 3, with the exception that: in the step (2), a tissue protective agent is not added, and the precipitated cells are obtained by directly centrifuging.
Comparative example 2-a method of in vitro expansion of limbal stem cells according to example 3, with the exception that: in the step (2), the tissue protective agent contains 3.0mg/ml beta-glucan and 3.0mg/ml L-glutamine.
Comparative example 3-a method of in vitro expansion of limbal stem cells according to example 3, with the exception that: in step (1), all limbal stem cell tissues are digested, and in step (3), only the resuspended cells are cultured.
1. In the in vitro limbal stem cell expansion methods according to examples 1 to 5 and comparative examples 1 to 3, the results of in vitro expansion culture of the same amount of limbal stem cells, observation of the morphological structure of the limbal stem cells cultured for 20 days, calculation of the cell viability of the precipitated cells and the cell expansion of primary and continuous 3-passage cells are shown in the following table,
Figure BDA0002711780410000061
as can be seen from the above table, the in vitro amplification methods of limbal stem cells in examples 1-5 of the present invention all have a survival rate of the precipitated cells of 98% or more, and the primary P cells thereof0And the cell expansion effect of the continuous passage 3 generations from P1 to P3 is stable, and the cell density of the continuous passage 3 generations can be kept at 9.0 multiplied by 105The above; compared with the embodiments 4 to 5, the embodiment 3 shows that the combination of a certain content of echinacoside and beta-glucan is beneficial to protecting the discrete limbal stem cells in the enzymolysis separation process, maintaining the cell proliferation activity of the limbal cells in the in vitro separation process and improving the cell proliferation stability. As can be seen from comparison between example 3 and comparative examples 1 to 3, the viability of the precipitated cells in comparative examples 1 and 2 is reduced, the stability of primary and subculture effects is low, the cell expansion capacity of P1-P3 in continuous passage 3 in comparative example 3 is obviously depleted, and the density of limbal stem cells is reduced to 4.9X 10 in P35The method has the advantages that on the basis of ensuring the full digestion of the limbal stem cells, the limbal stem cell basic culture solution DMEM containing a certain amount of echinacoside, beta-glucan and L-glutamine is used as a protective agent for centrifugal precipitation of cells, so that the effect of effectively protecting the discrete limbal stem cells in the enzymolysis separation process is achieved, the pollution of the limbal stem cells in vitro separation culture is reduced, the cell proliferation activity is better maintained in the in vitro separation process, and the stable proliferation activity of the cells is favorably maintained in the separation process. The comparative example 3 shows that the limbal stem cells which are combined and dispersed by trace undigested tissue blocks are also adopted for amplification culture under certain conditions, so that the limbal stem cells are favorably and better promotedThe growth and proliferation of the cells are facilitated, the film-forming growth of the cells is facilitated, the proliferation capacity is improved, and the characteristics of the limbal stem cells are maintained, so that the whole efficient and stable in-vitro amplification culture of the limbal stem cells is successfully realized, and the proliferation potential of the in-vitro amplification culture of the limbal stem cells is fully exerted.
2. The limbal stem cells of 3 generations of continuous passage in examples 1 to 5 and comparative examples 1 to 3 were subjected to immunochemical staining with antibodies for the specific markers PAX6, P63, K12 and Ki67 of the limbal stem cells of 3 generations of continuous passage, and blue color was DAPI and green color was developed with the antibodies, and the results showed that the limbal stem cells of 3 generations of continuous passage in examples and comparative examples of the present invention were all positive, had the characteristics of limbal stem cells, and the antibody positive rates were all 95% or more and were 100% at the maximum.
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 (8)

1. A method for in vitro expansion of limbal stem cells, comprising: the method comprises the following steps:
(1) cutting the limbal stem cell tissue blocks into pieces, flushing the limbal stem cell tissue blocks for 2-3 times by using Hank liquid in an aseptic environment, adding 30-50 times of digestive juice by volume into 90-95% of the limbal stem cell tissue by mass, digesting, and removing the digestive juice; after 2-3 times of flushing by Hank liquid, adding a limbus stem cell culture medium with the volume of 8-10 times, and stopping trypsinase digestion;
(2) adding a tissue protective agent with the volume 0.1-0.5 times that of the mixture, centrifuging for 8-10 min at the speed of 1000-1100 r/min, removing supernatant, and taking precipitated cells; the tissue protective agent is prepared by adopting a limbal stem cell basic culture solution DMEM and adding 0.55-1.25 ug/ml echinacoside, 1.5-2.5 mg/ml beta-glucan and 2.2-3.8 mg/ml L-glutamine for mixing;
(3) washing the precipitated cells with Hank solution for 1-2 times, adding the cells into a limbal stem cell culture medium with the volume of 3-5 times, re-suspending, mixing with 5-10% of undigested limbal stem cell tissue, planting the mixture into a culture plate with holes, and culturing at 36-37 DEG C93-95% humidity, 5-5.5% CO2Culturing in an incubator, and changing the culture solution every other day to obtain the limbal stem cells.
2. The method of claim 1, wherein the method comprises: in the step (1), before adding the limbal stem cell culture medium to terminate the trypsin digestion, adding a limbal stem cell basic culture solution DMEM/F12(1:1) containing 0.5-0.8% of fetal bovine serum, placing the mixture in a constant-temperature shaking table at 37 ℃ to react for 3-5 min, and sucking out the cell culture medium.
3. The method of claim 2, wherein the method comprises: the tissue protective agent contains 1.05ug/ml echinacoside, 2.0mg/ml beta-glucan and 3.0mg/ml L-glutamine.
4. The method of claim 1, wherein the method comprises: the limbal stem cell culture medium is prepared by mixing a limbal stem cell basic culture solution DMEM/F12(1:1) with 10-15% fetal bovine serum, 2.5-3 ml of 100 Xstreptomycin mixed solution, 0.3-0.5 mg/ml glutamine, 5-8 ug/ml insulin, 15-25 mg/ml transferrin, 0.2-0.5 ug/ml hydrocortisone, 10-16 ng/ml cholera toxin, 3-5 ng/ml platelet-derived factor, 1-3 ng/ml interleukin-6 and 10-15 ng/ml insulin-like growth factor.
5. The method of claim 1, wherein the method comprises: in the step (1), the digestive juice is obtained by mixing 1.25g/L of trypsin and 0.2g/L of EDTA1: 1.
6. The method of claim 1, wherein the method comprises: in the step (1), the consumption time is 13-15 min.
7. A limbal stem according to claim 1A method for in vitro amplification of cells, comprising: in step (3), the density of the inoculation of the resuspended cells in the well-containing culture plate is 1X 105Cells/cm2。
8. The method of claim 1, wherein the method comprises: in step (3), the tissue is resuspended in 4 volumes of limbal stem cell culture medium and mixed with 8% undigested limbal stem cell tissue and seeded into well-plated culture plates.
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