CN115869251A - Simple preparation method of retinal pigment epithelial cells - Google Patents
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- 238000002360 preparation method Methods 0.000 title abstract description 36
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- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
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Images
Abstract
The present application relates to a simple method and composition for retinal pigment epithelial cell preparation. Specifically, the application relates to preparation of a preparation, which contains sodium chloride injection, and the cell viability can be maintained to be more than 90% in 48 hours at the temperature of 2-8 ℃.
Description
Technical Field
The application relates to a preparation technology of a retinal pigment epithelial cell preparation, in particular to a preparation method of retinal pigment epithelial cells before clinical injection. The preparation method can be used for rapidly preparing the retinal pigment epithelial cell preparation, can ensure that the retinal pigment epithelial cell maintains the cell viability not less than 90% within at least 48 hours, and realizes the convenience of the retinal pigment epithelial cell in clinical application.
Background
Dry age-related macular degeneration (AMD) is caused by degeneration or damage of Retinal Pigment Epithelial (RPE) cells, and is one of the leading causes of blindness in the elderly, and, by incomplete statistics, this disease severely affects the quality of life of 3-5 million people worldwide and cannot be effectively treated by traditional pharmaceutical or surgical methods, and RPE cell transplantation has become a potential method for treating such diseases. In the process of RPE transplantation, the state and the survival rate of cells are ensured in each link of preparation, transportation, compatibility, injection and the like of RPE cells.
Disclosure of Invention
The inventor strives to develop an RPE preparation with simple operation and high cell survival rate through a large amount of research reasoning and experimental exploration. In practice and innovation for many years, the sodium chloride injection is found to well maintain the survival rate of RPE cells by adjusting the components of the culture medium for many times and combining the convenience of clinical use. Can provide the preparation type which can be directly injected without secondary operation for the clinical application of RPE cells.
Patent document CN104080464A relates to a pharmaceutical composition comprising a plurality of Retinal Pigment Epithelial (RPE) cells; and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier comprises a balanced salt solution of 7.14mg of sodium chloride, 0.38mg of potassium chloride, 0.154mg of calcium chloride dihydrate, 0.2mg of magnesium chloride hexahydrate, 0.42mg of disodium hydrogen phosphate, 2.1mg of sodium bicarbonate, 0.92mg of glucose, 0.184mg of glutathione disulfide (oxidized glutathione), and hydrochloric acid and/or sodium hydroxide (to adjust the pH to about 7.4) per mL of water. Although sodium chloride is also used in this patent, the composition of the solution is complicated, the solution preparation efficiency is low, and the economic cost is high. The simultaneous addition of additional ingredients may place greater demands on the tolerance of the additive to the animal or human to which the preparation is administered, and therefore it is desirable to add as little other components of the cell preparation as possible apart from the cells. However, maintaining cell viability is a major problem in the art. Therefore, there is an urgent need to develop an RPE preparation with simple components and high cell survival rate.
Generally, the RPE injection is prepared in the previous stage by the following method: after digestion of RPE cells into single cells, digestion was stopped with RPE medium, hank's Balanced Salt Solution (HBSS), phosphate buffered saline DPBS solution, and in preparation of RPE cell preparations, clinical injections were reported with RPE suspensions prepared with balanced salt solution BSS (Lancet 2012-379. The method also has the problems of more solution compositions, complex operation steps, high operation difficulty, high economic cost and the like.
In view of this, the present application provides a simpler combination of cell preparations and a method for preparing a cell preparation, which has an effect of increasing the survival rate of RPE cells.
The application provides an injection for preserving the survival rate of retinal pigment epithelial cells, which can maintain the cell survival rate of at least 70%, 75%, 80%, 85%, 90% and 95% at the temperature of 2-8 ℃.
In certain embodiments, the injection comprises sodium chloride.
In certain embodiments, the injection is capable of maintaining at least 70%, 75%, 80%, 85%, 90%, 95% cell viability for not less than 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours at 2-8 ℃.
In certain embodiments, the injection is capable of maintaining at least 70%, 75%, 80%, 85%, 90%, 95% cell viability over >0 ℃ for no less than 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours.
In certain embodiments, the injection is capable of maintaining at least 70%, 75%, 80%, 85%, 90%, 95% cell viability over 4-20 ℃ for no less than 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours.
In certain embodiments, the injection is capable of maintaining at least 70%, 75%, 80%, 85%, 90%, 95% cell viability over 20-30 ℃ for no less than 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours.
In certain embodiments, the injection is capable of maintaining at least 90% cell viability over a period of no less than 24 hours at 2-8 ℃.
In certain embodiments, the injection is capable of maintaining at least 90% cell viability over a period of no less than 48 hours at 2-8 ℃.
In certain embodiments, the injection is capable of maintaining at least 85% cell viability over a period of no less than 60 hours at 2-8 ℃.
In certain embodiments, the injection is capable of maintaining at least 90% cell viability over a period of no less than 24 hours at 18-25 ℃.
In certain embodiments, the injection is capable of maintaining at least 85% cell viability at 18-25 ℃ for no less than 48 hours.
In certain embodiments, the injection is capable of maintaining at least 80% cell viability for no less than 60 hours at 18-25 ℃.
In certain embodiments, the injection solution matches cell densities no less than 1x10 3 /mL, can be 1x10 4 /mL、1x10 5 /mL、1.5x10 5 /mL、2x10 5 /mL、2.5x10 5 /mL、1x10 6 /mL、1.5x10 6 /mL、2x10 6 /mL、3x10 6 /mL、1x10 7 and/mL, etc.
In some embodiments, the injection may also comprise one or more of compound sodium chloride, sodium bicarbonate injection, glucose sodium chloride injection and the like in any combination besides the sodium chloride injection.
The preparation method of the retinal pigment epithelial cell injection comprises the following steps:
digesting the RPE cells into single cells by using trypsin, collagenase and/or other enzymes, stopping digestion by using sodium chloride injection, collecting the cells into a centrifuge tube, and centrifuging at 800-2000rpm for 2-5 minutes; washing with sodium chloride injection for 2-5 times, and centrifuging at 800-2000rpm for 2-5 min; after washing, resuspending the cells with 0.9 mass% sodium chloride injection and counting;
adjusting the cell density by using sodium chloride injection according to the clinical cell density;
can be stored and transported at a temperature of >0 deg.C, optionally 2-8 deg.C, 1-4 deg.C, 4-20 deg.C or 20-30 deg.C.
In certain embodiments, the Retinal Pigment Epithelial (RPE) cells that can be used for preservation are derived from cells of mammalian triphase, such as, but not limited to, pluripotent stem cells (e.g., embryonic stem cells, induced pluripotent stem cells), functional cells derived from differentiation of pluripotent stem cells (e.g., retinal pigment epithelial cells, hepatocytes, cardiomyocytes, etc.), adult stem cells (e.g., umbilical cord-derived cells, adipose-derived cells, bone marrow-derived cells, neural stem cells, etc.), somatic cells (e.g., cardiomyocytes, fibroblasts, muscle cells, etc.).
The term "embryonic stem cell" herein is well known to those of ordinary skill in the art. The source may be a human or non-human animal. The human embryonic stem cells are limited to stem cells isolated from human embryos within 14 days of fertilization that have not undergone in vivo development.
The main prospects and meanings of the invention include:
1) Compared with the traditional RPE injection, the injection of the invention has simple components and convenient operation, reduces the preparation cost of the injection and improves the efficiency.
2) The injection of the invention has simple chemical components, but can obtain the RPE cell survival rate which is the same as or even better than other complex components, and the high survival rate provides good foundation for the drug use of retinal pigment epithelial cells and clinical trial drugs.
Description of the drawings:
FIG. 1 is a graph of cell inoculation after 24 hours of storage in sodium chloride injection at 2-8 ℃;
FIG. 2 is a graph of cell inoculation after 48 hours of preservation in sodium chloride injection at 2-8 ℃;
FIG. 3 is a graph of cell seeding after 12 hours storage in comparative experimental protection solutions 1-6 at 4 ℃.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the invention, its application, or uses. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
1) Preparation of RPE cell preparation
Digesting the RPE cells into single cells by using Tryple enzyme, stopping digestion by using sodium chloride injection, collecting the cells into a centrifuge tube, and centrifuging for 3 minutes at 1200 rpm;
washing with sodium chloride injection for 2-5 times, and centrifuging at 1200rpm for 3 min;
after washing, resuspending the cells with sodium chloride injection and counting;
adjusting the cell density by sodium chloride injection according to the clinical cell density;
storing at 2-8 deg.C, and transporting.
2) Determination of Activity
The RPE cell preparations prepared in example 1 were subjected to cell viability measurement at the time of 24 hours and 48 hours of preservation while the cells were seeded into a culture plate so as to observe the cell state.
The method for testing the experimental samples (the test results are shown in table 1) is as follows:
the RPE preparation prepared by the sodium chloride injection is preserved for 24 hours at the temperature of 2-8 ℃, and the cell survival rate is determined to be 92.3 percent by counting the cell survival rate; preserving the RPE preparation prepared by the sodium chloride injection for 48 hours at the temperature of 2-8 ℃, and determining the cell survival rate to be 91% by counting the cell survival rate; the RPE preparation prepared by the sodium chloride injection is preserved for 24 hours at the temperature of 18-25 ℃, and the cell survival rate is determined to be 92.2 percent by counting the cell survival rate; the RPE preparation prepared by the sodium chloride injection is preserved for 48 hours at the temperature of 18-25 ℃, and the cell survival rate is determined to be 85.4 percent by counting the cell survival rate.
In addition, the RPE preparation prepared by the sodium chloride injection is preserved for 60 hours at the temperature of 2-8 ℃, and the cell survival rate is determined to be 89% by cell survival rate counting; the RPE preparation prepared by the sodium chloride injection is preserved for 60 hours at the temperature of 18-25 ℃, and the cell survival rate is determined to be 85.0 percent by counting the cell survival rate. The results are shown in Table 1.
TABLE 1 statistics of cell viability of injections at different temperatures and different storage times
3) Observation of cell State
Vitronetin was diluted with 4 ℃ pre-cooled DPBS-CTS, 1ml of diluted matrigel was added to each of six well plates, and the plates were placed in an incubator at 37 ℃ for 1 hour.
The RPE preparation was transferred to a 15mL centrifuge tube and centrifuged at 1200rpm/min for 3min.
The supernatant was discarded, and the corresponding volume of cell culture medium was added and mixed well. Adding into the coated board. The cell culture plate is put back into the carbon dioxide incubator, after shaking evenly, the incubator door is lightly closed.
The adherent growth of the cells was observed the next day.
The RPE preparation prepared by the sodium chloride injection is preserved for 24 hours at the temperature of 2-8 ℃ and then cell inoculation is carried out. As shown in fig. 1: essentially all cells were adherent and the cells grew well with very few cells unattached.
The RPE preparation prepared by the sodium chloride injection is preserved for 48 hours at the temperature of 2-8 ℃ and then cell inoculation is carried out. As shown in fig. 2: essentially all cells were adherent and the cells grew well with very few cells unattached.
4) Comparative example 1 experiment with other injection solutions
After the RPE cells had digested into single cells, the digestion was terminated with the injections shown in Table 2, and the digested RPE cells were stored in six injections shown in Table 2 at 4 ℃ for 12 hours, respectively, before cell inoculation. Wherein EB means a culture solution of RPE. With reference to the method of example 1, adherent growth of cells was observed as shown in FIG. 3. The results show that non-adherent cells are more pronounced compared to example 1. Nonadherent cells in the supernatant were collected, counted and the results are listed in the table below. It can be seen that the survival rate of the cells stored in the protective solutions 1 to 6 for 12 hours (the highest rate of 91.1%) is significantly lower than that of the cells stored in the protective solutions in example 1 for 24 hours (both rates are more than 92%). The improvement of the survival rate of 1% has great significance in clinical treatment, and compared with the six injections shown in table 2, the sodium chloride injection in the embodiment 1 has the advantages of low economic cost and direct in vivo injection, and improves the use convenience of the RPE cell preparation.
TABLE 2 LIGHTEN DEADJACENT CELL LIST OF SIX INJECTIONS
| Serial number | Name of injection | Proportion of non-adherent cells | Proportion of adherent viable cells |
| 1 | DPBS | 8.9％ | 91.1％ |
| 2 | DPBS+Y27632 | 9.0％ | 91％ |
| 3 | HBSS | 10.2％ | 89.8％ |
| 4 | HBSS+Y27632 | 10％ | 90％ |
| 5 | EB | 11.2％ | 88.8％ |
| 6 | EB+Y27632 | 12.1％ | 87.9％ |
Claims (9)
1. An injection with high survival rate of retinal pigment epithelial cells can maintain the cell survival rate of more than 80 percent under the condition of not less than 24 hours.
2. The injection solution of claim 1, wherein the retinal pigment epithelial cells and the components other than the sodium chloride injection are not included.
3. The injection solution of any one of claims 1-2, which can maintain at least 80%, 85%, 90%, 95% of cell viability within 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours at 1-8 ℃.
4. The injection solution of any one of claims 1-2, which can maintain at least 80%, 85%, 90%, 95% of cell viability within 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours at 4-20 ℃.
5. The injection solution of any one of claims 1-2, which can maintain cell viability of at least 80%, 85%, 90%, 95% in 20-30 ℃ for not less than 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours.
6. The injection according to claim 1 to 5, wherein the cell density of retinal pigment epithelial cells is not less than 1x10 3 /mL。
7. The injection of any one of claims 1 or 3-6, which comprises retinal pigment epithelial cells and sodium chloride, and can also comprise one or any combination of compound sodium chloride, sodium bicarbonate injection and glucose sodium chloride injection.
8. The injection according to any one of claims 1 to 7, wherein the cell is derived from a mammalian three-germ layer cell, such as a pluripotent stem cell, a functional cell derived from differentiation of a pluripotent stem cell, an adult stem cell, or a somatic cell.
9. The injection according to any one of claims 1 to 8, characterized in that it is prepared by the following method:
digesting the RPE cells into single cells, stopping digestion by using sodium chloride injection, and centrifuging the cells; washing with sodium chloride injection, finally centrifuging again, removing supernatant, retaining cell precipitate, and then resuspending cells with sodium chloride injection.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1966080A (en) * | 2005-11-17 | 2007-05-23 | 北京科宇联合干细胞生物技术有限公司 | Neural stem cell injection for treating senile dementia and Parkinson's disease |
| CN112544612A (en) * | 2020-12-18 | 2021-03-26 | 江苏艾尔康生物医药科技有限公司 | Frozen stock solution and application thereof in RPE cell freezing |
-
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- 2021-09-27 CN CN202111136904.3A patent/CN115869251A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1966080A (en) * | 2005-11-17 | 2007-05-23 | 北京科宇联合干细胞生物技术有限公司 | Neural stem cell injection for treating senile dementia and Parkinson's disease |
| CN112544612A (en) * | 2020-12-18 | 2021-03-26 | 江苏艾尔康生物医药科技有限公司 | Frozen stock solution and application thereof in RPE cell freezing |
Non-Patent Citations (3)
| Title |
|---|
| EVA D. UEBERSAX等: "Survival of the Retinal Pigment Epithelium in vitro: Comparison of Freshly Isolated and Subcultured Cells", 《EXP. EYE RES.》, 31 December 2000 (2000-12-31), pages 381 - 390 * |
| 王璟等: "蒺藜总皂苷对缺血再灌注损伤大鼠视网膜神经细胞凋亡的影响", 《湖北科技学院学报（医学版）》, vol. 29, no. 3, 31 December 2015 (2015-12-31), pages 194 - 196 * |
| 纳涛等: "人胚胎干细胞来源的视网膜色素上皮细胞质量控制研究", 《生命科学》, vol. 30, no. 3, 31 March 2018 (2018-03-31), pages 248 - 260 * |
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