CN115216445A - Culture medium and application thereof in culturing retinal progenitor cells - Google Patents
Culture medium and application thereof in culturing retinal progenitor cells Download PDFInfo
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Abstract
The invention relates to the technical field of cell biology, in particular to a culture medium and application thereof in culturing retinal progenitor cells. The culture medium provided by the invention is suitable for the growth of the retinal progenitor cells under the hypoxic condition, can maintain the culture under the hypoxic condition and can continuously maintain the dryness. And the retinal progenitor cells cultured by the culture medium still have the capacity of differentiating into various types of downstream retinal constitutional cells, are less differentiated into glial cells, and can secrete a large amount of neuroprotective factors. Under the condition of hypoxia, the cell can be expanded to P20 generation by using the culture medium, the cell characteristics are maintained, and the yield is improved by more than 40 times compared with other commercial culture media. And the liquid changing frequency and the liquid changing amount can be reduced, the retina physiological oxygen partial pressure environment can be more effectively simulated, the influence of active oxygen free radicals on cells is reduced, the instability caused by frequent operation is reduced, stable and uniform retina progenitor cells in batches can be produced, and the industrialization is more facilitated.
Description
Technical Field
The invention relates to the technical field of cell biology, in particular to a culture medium and application thereof in culturing retinal progenitor cells.
Background
Retinal Progenitor Cells (RPCs) are a class of tissue-specific stem Cells located in Retinal tissues that can specifically differentiate into mature Retinal functional Cells, and are the basis for the formation and functional exertion of Retinal Cells. Theoretically, adult retinal cells cannot regenerate after being damaged, so that for a patient with retinopathy, the retinal progenitor cells cultured in vitro and transplanted to a pathological change part can intervene or delay the progression of retinopathy, improve the vision of the patient and improve the functions of the retinal cells. At present, a plurality of clinical trial researches have been carried out to transplant the retinal progenitor cells cultured in vitro into the eyes of patients with retinitis pigmentosa, and all the researches report that the retinal progenitor cells transplanted into the eyes are safe, and the eyesight of the patients can be obviously improved and the living conditions of the patients can be improved after the treatment. Thus, retinal progenitor cells cultured in vitro provide a novel approach to the treatment of retinal degenerative diseases. Most of the existing culture systems of the retinal progenitor cells and the neural stem cells are cultured under an normoxic condition, on one hand, the cells generate energy by utilizing glycometabolism and simultaneously generate ATP energy by applying oxidative phosphorylation under the normoxic condition, and a large amount of active oxygen free radicals can be generated by long-term normal oxygen partial pressure culture in vitro to damage the stem cells, so that the non-directional differentiation and the apoptosis of the stem cells are caused.
Retinal progenitor cell transplantation is one of the under-developed therapies for treating retinal degeneration diseases, the retinal progenitor cells are isolated from the retinal tissue of a developing fetal eye, the content of the retinal progenitor cells is low, the in vitro amplification capacity is limited, therefore, in order to improve the amplification efficiency of the retinal progenitor cells in vitro, a great amount of research is carried out on the retinal progenitor cells amplified by using culture conditions simulating the physiological oxygen partial pressure conditions of the retinal tissue in the developing stage, although the method can achieve the aim of in vitro amplification of the retinal progenitor cells, the improvement effect on the cell yield is limited, most of the reported culture media still adopt a normal oxygen partial pressure scheme, and the problem of cell energy metabolism conversion under the hypoxia condition is not considered, so the method is not suitable for culturing the cells under the hypoxia condition, or needs to frequently replace fresh culture media to maintain the energy required for cell growth, and the frequent hypoxia and reoxygenation of the cells during the solution replacement process causes instability of the cells, and is easy to cause stem cell damage.
In the prior art, the retinal progenitor cells and the neural stem cells under the hypoxia condition are cultured by adopting an normoxic culture medium, and the requirement of cell energy metabolism change under the hypoxia condition is not considered, so that the conventional culture medium is not suitable for the requirement of retinal progenitor cell growth under the hypoxia condition, and the cell damage condition can be increased by frequent reoxygenation-hypoxia liquid exchange operation, thereby reducing the treatment effect. Therefore, there is an urgent need to improve the expansion effect of retinal progenitor cells and neural stem cells and maintain the dryness of cells during the expansion process.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a culture medium and its application in culturing retinal progenitor cells. Aiming at the characteristic of cell energy metabolism under hypoxia, the invention innovatively invents a culture system meeting the requirement of hypoxia culture, and meanwhile, the culture system can also be applied to the culture of neural stem cells and retinal progenitor cells under the normoxic condition, so that the influence of active oxygen free radicals on the cells under the normoxic condition can be reduced, and the cells are protected from being damaged.
The present invention provides a composition comprising: EGF, bFGF, glutaMAX-1, rapamycin, PS48, UK5099, and 1,6 fructose diphosphate. The influence of the lack of factors on cell proliferation is tried in the construction process of the culture medium, and finally, the combination of the factors is optimally found to be most vigorous on cell proliferation.
The composition provided by the invention is characterized in that the mass ratio of EGF, bFGF, glutaMAX-1, rapamycin, PS48, UK5099 and 1,6-fructose diphosphate is 1: 2: 3000000: 100: 1000: 500: 50000000.
The invention provides a culture medium, which comprises a basic culture medium and the composition.
The culture medium comprises a basic culture medium, EGF 1 ng/mL-50 ng/mL, bFGF 1 ng/mL-50 ng/mL, glutaMAX-1 3mg/mL-30 mg/mL, rapamycin 0.1 mu g/mL-1 mu g/mL, PS 48.5 mu g/mL-20 mu g/mL, UK 5099.5 mu g/mL-5 mu g/mL and 1,6-fructose diphosphate 100 mg/mL-1 g/mL.
In some embodiments, the medium consists of a basal medium and EGF 5 ng/mL-30 ng/mL, bFGF 5 ng/mL-30 ng/mL, glutaMAX-1 10mg/mL-20 mg/mL, rapamycin 0.5 μ g/mL-1 μ g/mL, PS48 μ g/mL-10 μ g/mL, UK50991 μ g/mL-5 μ g/mL, and 1,6-fructose diphosphate 100 mg/mL-50 mg/mL.
In other embodiments, the medium consists of basal medium and EGF 10ng/mL, bFGF 20ng/mL, GIutaMAX-1 30mg/mL, rapamycin 1. Mu.g/mL, PS 48. Mu.g/mL, UK 5099. Mu.g/mL, and 1,6-fructose diphosphate 500mg/mL.
The culture medium provided by the invention is characterized in that the basic culture medium is Advanced DMFM/F12 culture medium.
In the culture medium provided by the invention, EGF, bFGF, glutaMAX-1, rapamycin, PS48, UK5099 and 1,6-fructose diphosphate are mutually coordinated, and each component is one-to-one deficient. Previous experiments showed that the replacement or reduction of any of the components resulted in a substantial reduction in cell culture. Moreover, the matching ratio of each component also has a significant influence. The culture medium of the invention can be used for culturing the retinal progenitor cells, so that the retinal progenitor cells can be efficiently expanded until the P20 generation does not have senescence, and the dryness and the directional differentiation capability of the cells can be still maintained. Furthermore, the retinal progenitor cells cultured by the culture medium obviously reduce the differentiation towards GFAP (glial fibrin protein) cells, reduce the generation of GFAP positive glial cells, more preferentially differentiate towards functional cells, and express at least one of the neuroprotective factors GFBP-2, IGFBP-3, IGFBP-4, BDNF, GDF-15, FGF-4, FGF-7 and/or PDGF at a high level. Wherein the factors such as IGFBP-3, BDNF, FGF-4, FGF-7, PDGF and the like are improved more obviously.
Moreover, the retinal progenitor cells cultured by the culture medium highly express apoptosis suppressor genes, and bcl2, p21, CDK2 and CDK1 prove that the culture medium has a protective effect on the retinal progenitor cells.
The invention provides a method for culturing retinal progenitor cells, which comprises the steps of culturing the cells by using the culture medium at the oxygen concentration of 1-6.5% and carrying out passage.
In some embodiments, the method comprises culturing cells using the medium at an oxygen concentration of 2% to 6% and passaging.
In other embodiments, the method comprises culturing the cells and passaging with the medium at an oxygen concentration of 2%, 3%,4%, 5%, 6%, respectively.
In some embodiments, the method comprises culturing cells using the medium at an oxygen concentration of 5% and passaging.
In the method for culturing retinal progenitor cells, the seeding density is 9,000cells/cm 2 ～13,000cells/cm 2 The culture medium is replaced every 2 days, the culture medium is replaced every 4 days, and CO is added 2 The concentration is controlled to be 5%, most cells can be seen to adhere to the wall 24h after inoculation, the cells are observed to be single under a phase contrast microscope, the cell bodies are small and transparent, and a few of the protrusions extend out. And starting cell subculture amplification after the cells are more than 85% confluent.
The culture medium can also be used for culturing the retinal progenitor cells at the oxygen concentration of 20%, and can effectively reduce the generation of oxygen free radicals ROS and protect the cells from being damaged.
The retinal progenitor cells cultured by the method still have the capacity of differentiating into retinal constitutional cells, and the glial cells with low GFAP positive expression preferentially express retinal photoreceptor cells marker and retinal dry marker, so that the retinal photoreceptor cells are favorably differentiated.
The invention also provides the retinal progenitor cells obtained by the culture of the method.
The retinal progenitor cells cultured by the method of the invention express neuroprotective factors at high levels. The neuroprotective factors include: at least one of GFBP-2, IGFBP-3, IGFBP-4, BDNF, GDF-15, FGF-4, FGF-7 and/or PDGF. Compared with the prior art, the neuroprotective factors in the retinal progenitor cells obtained by the culture of the invention are remarkably improved, wherein the improvement of IGFBP-3, BDNF, FGF-4, FGF-7 and PDGF is more remarkable, and the concentration of each factor in the culture system of the invention can reach IGFBP-3:1206.03pg/ml, BDNF:728.1pg/ml, FGF-4:910.3pg/ml, FGF-7:758.4pg/ml, PDGF:961.967pg/ml. And the retinal progenitor cells cultured by the method of the invention have low expression of apoptosis promoting genes.
The invention provides application of the retinal progenitor cells in preparing products for treating and/or preventing diseases related to retinal nerve cell apoptosis.
In the application, the retinal nerve cell apoptosis related diseases comprise retinitis pigmentosa, diabetic retinopathy and/or age-related macular degeneration.
The invention also provides a product for treating and/or preventing diseases related to apoptosis of retinal nerve cells, which comprises the retinal progenitor cells obtained by the culture of the method.
The product of the invention is a cell preparation containing retinal progenitor cells. It is a cell suspension or a cell freeze-dried powder, and comprises a reagent of the retina progenitor cells and suspension cells, or comprises a freeze-drying protective agent. Other drugs for treating and/or preventing diseases related to retinal nerve cell apoptosis can also be included.
The invention also provides a method for treating and/or preventing diseases related to retinal nerve cell apoptosis, which comprises the step of administering the product.
The invention provides a culture medium, which is more suitable for the growth of retinal progenitor cells under the condition of hypoxia, can maintain the long-term culture of the retinal progenitor cells under the condition of hypoxia, can continuously maintain the dryness of the retinal progenitor cells, and the retinal progenitor cells cultured by the culture medium still have the capacity of differentiating into various types of downstream retinal constitutional cells, are less differentiated into glial cells, and can secrete a larger amount of neuroprotective factors. Experiments prove that under the hypoxia condition, the cell can be expanded to P20 generation in vitro by using the culture medium, the characteristics of the retinal progenitor cell are still maintained, and the yield of the cell is improved by more than 40 times compared with other commercial culture media. The method can reduce the liquid changing times and the liquid changing amount, more effectively simulate the physiological oxygen partial pressure environment of the retina, and reduce the influence of active oxygen free radicals on cells, thereby further improving the yield of the retinal progenitor cells, and reducing the instability of the cells caused by frequent operation.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts according to the drawings:
FIG. 1 shows a comparison of the proliferative capacity of retinal progenitor cells at 5% oxygen concentration with different combinations of factors;
FIG. 2 shows a comparison of the multiplication capacity of retinal progenitor cells in different generations in different culture systems at 5% oxygen concentration;
FIG. 3 shows comparative analysis of retinal progenitor cell cycle at 5% oxygen concentration in different culture systems;
FIG. 4 shows the changes in expression of pro-and apoptosis-related genes by retinal progenitor cells in different culture systems at 5% oxygen concentration;
FIG. 5 shows the expression of dry markers of retinal progenitor cells in different culture systems at 5% oxygen concentration;
FIG. 6 shows a comparison of the differentiation capacity of retinal progenitor cells under differentiation inducing conditions at an oxygen concentration of 5%;
FIG. 7 shows that retinal progenitor cells secrete neuroprotective factors at 5% oxygen concentration under different culture systems.
Detailed Description
The invention provides a culture medium and application thereof in culturing retinal progenitor cells. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1 establishment of hypoxic culture System for human retinal progenitor cells
1. Establishment of isolation culture system of primary human retinal progenitor cells
(1) Pre-culture container matrix glue Cell start coating: the matrigel Cell start contains Ca according to the proportion of 1: 50 2+ 、Mg 2+ Is diluted and added to the flask, 2 ml/flask, 5% CO 2 IncubatorCoating at 37 deg.C for 1h or overnight at 4 deg.C;
(2) Separating intact retina tissue of eye under dissecting microscope, transferring to DMEM culture solution at 4 deg.C, and rinsing for 2 times;
(3) Transfer of retinal tissue to a tissue containing 1ml TrypLE TM -Express digest, vortexed every 5 minutes at 37 ℃ until it appears as a single cell suspension;
(4) Adding 3ml of KOSR medium containing 5% of the total weight of the cells to terminate the digestion, gently blowing the cell suspension, centrifuging for 1000 rpm for 3 minutes, and discarding the supernatant;
(5) Adding 5ml of normal oxygen culture medium (ULTRACULTURE) respectively TM Medium, EGF 10ng/mL;20ng/mL bFGF;1% GlutaMAX-1) and 5mL of the medium of the invention (Advanced DMFM/F12 medium, EGF 10ng/mL;20ng/mL bFGF;1% GlutaMAX-1; rapamycin 0.1nM; PS 48. Mu.M; 0.5mm UK5099;1,6-Fructose Diphosphate (FDP) 50 mM), cell count, and seeding into flasks previously coated with cellstar. The cell density was 9000cells/cm 2 ～13000cells/cm 2 Adding 5ml of 5% KOSR medium, placing in a low-oxygen culture chamber, and adding CO 2 Concentration was controlled to 5%, O 2 The concentration is controlled to be 5 percent, and the temperature is 37 ℃; the culture medium has the effect of the lack of the test factors on cell proliferation in the construction process, and finally, the combination of the factors is optimally found to have the most vigorous cell proliferation.
(6) After 24h of inoculation, most cells can be seen attached to the wall, and the cells are observed to be single under a phase contrast microscope, the cell bodies are small and transparent, and a few protrusions extend out. The culture medium needs to be replaced every 2 days for the normoxic culture medium, the culture medium is replaced every 4 days for the invention patent culture medium, and cell subculture amplification culture is started after more than 85% of cells are converged.
2. Hypoxia culture system for promoting proliferation of human retinal progenitor cells
Human retinal progenitor cells under hypoxic conditions (5%O) 2 Concentration), after culturing the culture medium and the normal oxygen culture medium, taking the cells with good growth state to prepare a cell suspension with a certain concentration, and adding 2 multiplied by 10 into each hole 3 Cells were added to a 96-well cell culture plate at 100. Mu.l/100. Mu.l well. Detecting increase in cellsReproductive capacity, doubling time, cell cycle and expression changes of cell apoptosis promoting and apoptosis inhibiting related genes. The results are shown in FIGS. 1 to 5.
As shown in fig. 1: in the process of constructing the culture medium, the proliferation capacity of the retinal progenitor cells is analyzed through the comparison of different factor combinations, and as can be seen from figure 1, after the retinal progenitor cells are cultured for 72 hours and added with exogenous factors, the proliferation capacity of the retinal progenitor cells is remarkably improved compared with that of a conventional culture medium (a basic combination of EGF + bFGF + Glu containing three factors), wherein the seven combined factor culture media provided by the invention have the strongest cell proliferation promoting capacity. * Represents P < 0.05, represents P < 0.01, represents P < 0.001, represents P < 0.0001.
As shown in fig. 2: at 5%O 2 Cells cultured with the culture medium of this patent at concentrations can expand to P20 passages, whereas normoxic medium expands only to P10 passages under these conditions. The ordinate represents doubling time, and the abscissa represents passage number.
As shown in fig. 3: it can be seen that the retinal progenitor cells cultured under the inventive culture medium system have a significant increase in S phase and a decrease in G1 phase, indicating that most cells are in a proliferative state.
As shown in fig. 4: retinal progenitor cells at 5%O 2 The difference between the expression of the apoptosis-promoting genes (Caspase-3, caspase-9, caspase-7, bax) and the expression of the apoptosis-inhibiting genes (Bcl 2, P21, CDK2, CDK 1) in the two culture media under the culture condition can be seen, and the culture media disclosed by the invention can obviously promote the cells to express the apoptosis-inhibiting genes and can lowly express the apoptosis-promoting genes. The culture medium of the invention is proved to be capable of promoting cell proliferation and inhibiting cell apoptosis. * Represents P < 0.05, represents P < 0.01, and represents P < 0.001.
As shown in fig. 5: for the culture at 5%O 2 Concentration of retinal progenitors for fluorescence staining of sternness-specific marker proteins, nestin: neural stem cell-specific marker protein, sox2: a neural stem cell marker protein; pax6: retinal progenitor cell-specific marker protein, ki67: a cell proliferation marker protein. From the results of statistical analysis, it can be seen that retinal progenitor cells cultured using the patented medium canThe retinal progenitor cell specific marker protein Pax6 is highly expressed, and the expression of a proliferation marker Ki-67 is also obviously improved. From the statistical figures, nestin and Sox2 expression were slightly elevated, with no significant difference.
3. Hypoxia culture system for maintaining dryness and induced related protein expression of human retinal progenitor cells
The result shows that the retinal progenitor cells cultured by the method still have the capacity of differentiating into retinal constitutional cells, and the glial cells with low expression GFAP positive preferentially express retinal photoreceptor cells marker and retinal dry marker. The results are shown in FIG. 6: it can be seen that at 5%O 2 Under the condition, the retinal progenitor cells still have the capability of multidirectional differentiation, and under the culture system disclosed by the invention, the retinal progenitor cells are obviously reduced to be differentiated into retinal glial cells, and more to be differentiated into functional cells. GFAP: a retinal glial cell marker protein; map2: a mature retinal neuron marker; NF: a retinal neural tubulin marker; recoverin: a retinal photoreceptor cell marker; rhodopsin: the retinal rhodopsin marker, the number of which reflects the number of retinal rod cells.
4. Hypoxia culture system for promoting human retinal progenitor cells to secrete neurotrophic factors
The ELISA kit is used for detecting the capability of the retinal progenitor cells to secrete factors under different culture systems, and the detection result shows that the cells secrete more IGFBP-3, BDNF, FGF-4, FGF-7 and PDGF in the culture medium. The results are shown in FIG. 7: the retinal progenitor cells are cultured under physiological oxygen partial pressure, and the difference of cytokine expression is compared under different culture medium culture conditions, and the graph can see that: IGFBP-3, BDNF, FGF-4, FGF-7 and PDGF expression are obviously improved.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and these modifications or substitutions do not depart from the spirit of the corresponding technical solutions of the embodiments of the present invention.
Claims (10)
1. A composition, characterized in that the composition comprises: EGF, bFGF, glutaMAX-1, rapamycin, PS48, UK5099, and 1,6 fructose diphosphate.
2. The composition of claim 1, wherein the mass ratio of EGF, bFGF, glutaMAX-1, rapamycin, PS48, UK5099, and 1,6-fructose diphosphate is 1: 2: 3000000: 100: 1000: 500: 50000000.
3. A culture medium comprising a basal medium and the composition of claim 1 or 2.
4. The culture medium according to claim 3, characterized in that it consists of basal medium and EGF 1 ng/mL-50 ng/mL, bFGF 1 ng/mL-50 ng/mL, glutaMAX-1 3mg/mL-30 mg/mL, rapamycin 0.1 μ g/mL-1 μ g/mL, PS 48.5 μ g/mL-20 μ g/mL, UK 5099.5 μ g/mL-5 μ g/mL and 1,6-fructose diphosphate 100 mg/mL-1 g/mL.
5. The culture medium according to claim 3 or 4, wherein the basal medium is Advanced DMFM/F12 medium.
6. A method for culturing retinal progenitor cells, which comprises culturing the cells in the medium according to any one of claims 3 to 5 at an oxygen concentration of 1% to 6.5% and passaging the cells.
7. Retinal progenitor cells obtained by culturing according to the method of claim 6.
8. The retinal progenitor cell according to claim 7, characterized in that it expresses: IGFBP-2, IGFBP-3, IGFBP-4, BDNF, GDF-15, FGF-4, FGF-7 and/or PDGF.
9. Use of a retinal progenitor cell according to claim 7 or 8 for the preparation of a product for the treatment and/or prevention of a disease associated with apoptosis of retinal nerve cells.
10. A product for the treatment and/or prevention of a disease associated with apoptosis of retinal nerve cells, said product comprising retinal progenitor cells according to claim 7 or 8.
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| CN102747029A (en) * | 2012-07-30 | 2012-10-24 | 何伟 | Culture method for retina progenitor cells and culture medium thereof |
| US20140186309A1 (en) * | 2011-05-18 | 2014-07-03 | The Regents Of The University Of California | Compositions and methods for treating retinal diseases |
| CN106318909A (en) * | 2015-06-23 | 2017-01-11 | 何伟 | Retinal progenitor cell and preparation thereof having function of treating degenerative retinal diseases |
| CN106323845A (en) * | 2015-06-23 | 2017-01-11 | 何伟 | Method and kit for identifying retinal progenitor cells capable of treating retinal degenerative change |
| CN113348243A (en) * | 2018-09-27 | 2021-09-03 | 加利福尼亚大学董事会 | Method for separating and culturing human retinal progenitor cells |
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| US20140186309A1 (en) * | 2011-05-18 | 2014-07-03 | The Regents Of The University Of California | Compositions and methods for treating retinal diseases |
| CN102747029A (en) * | 2012-07-30 | 2012-10-24 | 何伟 | Culture method for retina progenitor cells and culture medium thereof |
| CN106318909A (en) * | 2015-06-23 | 2017-01-11 | 何伟 | Retinal progenitor cell and preparation thereof having function of treating degenerative retinal diseases |
| CN106323845A (en) * | 2015-06-23 | 2017-01-11 | 何伟 | Method and kit for identifying retinal progenitor cells capable of treating retinal degenerative change |
| CN113348243A (en) * | 2018-09-27 | 2021-09-03 | 加利福尼亚大学董事会 | Method for separating and culturing human retinal progenitor cells |
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