Human endothelial progenitor cell culture medium and application thereof in preparation of extracellular vesicles

Abstract

The invention discloses a human endothelial progenitor cell culture medium and application thereof in preparation of extracellular vesicles. The formula of the human endothelial progenitor cell culture medium is as follows: human platelet lysate with the volume concentration of 3-10%, VEGF (vascular endothelial growth factor) with the volume concentration of 60-200ng / mL, IGF (insulin-like growth factor) with the volume concentration of 20-100ng / mL, L-glutamine with the volume concentration of 2-10mM, sodium pyruvate with the volume concentration of 0.02-0.75 mM, glucocorticoid with the volume concentration of 50-250ng / mL and a reducing agent with the volume concentration of 50-200mu M are added into a basic cell culture medium; the human endothelial progenitor cells are human placenta tissue-derived endothelial progenitor cells. Animal-derived reagents are not involved, the price is low, the number of EPCs extracellular vesicles obtained through amplification is large, the purity is high, and the skin wound repairing effect is good. The extracellular vesicles are easy to produce on a large scale, convenient to store and control batch quality, simple to obtain and use and free of immunological rejection.

Description

Technical Field

[0001] The present invention belongs to the technical field of human endothelial progenitor cell culture media, and particularly relates to a human endothelial progenitor cell culture medium and its application in the preparation of extracellular vesicles. Background Art

[0002] Extracellular vesicles (EVs) are nanoscale particles secreted by cells, which are cystic vesicles with a lipid membrane structure. By carrying and transmitting functional molecules such as RNA, proteins, and metabolites, they mediate the interaction between the environment and organisms, and between organisms, and have become signal communication carriers that have received wide attention.

[0003] Endothelial progenitor cells (EPCs) are precursor cells of endothelial cells and participate in tissue damage repair. EPCs from different sources have different functions. The sources of human EPCs are limited, there may be certain immune rejection for EPCs from different tissue sources, and it is difficult to maintain cell activity, which restricts the clinical use of EPCs. Moreover, it is difficult to produce on a large scale and is not easy to store.

[0004] Finding a new, scalable method for culturing human endothelial progenitor cells and expanding their clinical applications is a technical problem to be solved. Summary of the Invention

[0005] The purpose of this part is to outline some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments.

[0006] As one aspect of the present invention, the present invention provides a human endothelial progenitor cell culture medium, wherein: the formula of the human endothelial progenitor cell culture medium is: adding 3-10% (v / v) human platelet lysate, 60-200 ng / mL VEGF, 20-100 ng / mL IGF, 2-10 mM L-glutamine, 0.02-0.75 mM sodium pyruvate, 50-250 ng / mL glucocorticoid, and 50-200 μM reducing agent to a cell basal medium; the human endothelial progenitor cells are endothelial progenitor cells derived from human placenta tissue.

[0007] As a preferred scheme of the human endothelial progenitor cell culture medium of the present invention: the glucocorticoid includes one or more of hydrocortisone, cortisone acetate, and prednisone; the reducing agent includes one or more of ascorbic acid, cysteine, and sodium sulfite.

[0008] As a preferred embodiment of the human endothelial progenitor cell medium of the present invention: The formulation of the human endothelial progenitor cell medium is as follows: Add 5% by volume of human platelet lysate, 100 ng / mL of VEGF, 30 ng / mL of IGF, 6.5 mM of L-glutamine, 0.5 mM of sodium pyruvate, 100 ng / mL of hydrocortisone, and 100 μM of ascorbic acid to the basal cell medium.

[0009] The present invention also discloses the application of the human endothelial progenitor cell medium in the preparation of extracellular vesicles, wherein: the human endothelial progenitor cells are endothelial progenitor cells derived from human placental tissue, and the extracellular vesicles of the endothelial progenitor cells derived from human placental tissue can be used for the preparation of skin wound repair drugs.

[0010] As a preferred embodiment of the application of the human endothelial progenitor cell medium of the present invention in the preparation of extracellular vesicles: The method for preparing the extracellular vesicles of the endothelial progenitor cells derived from human placental tissue includes differential centrifugation.

[0011] As a preferred embodiment of the application of the human endothelial progenitor cell medium of the present invention in the preparation of extracellular vesicles: The method for preparing the extracellular vesicles of the endothelial progenitor cells derived from human placental tissue is as follows: Culture the endothelial progenitor cells derived from placental tissue with the human endothelial progenitor cell medium, collect the supernatant of the medium, centrifuge at 300 g for 10 min; take the supernatant and centrifuge at 2000 g for 10 min; take the supernatant and centrifuge at 10000 g for 30 min; take the supernatant and centrifuge at 100000 g for 80 min; discard the supernatant to obtain the extracellular vesicles.

[0012] As a preferred embodiment of the application of the human endothelial progenitor cell medium of the present invention in the preparation of extracellular vesicles: The endothelial progenitor cells derived from human placental tissue are obtained by adding trypsin and collagenase to digest placental tissue, separating mononuclear cells using lymphocyte separation medium, and inducing primary endothelial progenitor cells with this medium.

[0013] As a preferred embodiment of the application of the human endothelial progenitor cell medium of the present invention in the preparation of extracellular vesicles: It also includes passaging the endothelial progenitor cells derived from human placental tissue to obtain passaged endothelial progenitor cells.

[0014] As a preferred embodiment of the application of the human endothelial progenitor cell medium of the present invention in the preparation of extracellular vesicles: The passaging includes changing the cell medium every 48 - 72 hours.

[0015] As a preferred embodiment of the application of the human endothelial progenitor cell medium of the present invention in the preparation of extracellular vesicles: The endothelial progenitor cells are passaged 2 - 3 days after culturing.

[0016] Advantages of the present invention:

[0017] The proliferation rate of EPCs cells cultured with the cell culture medium of the present invention is significantly increased, and the yield of extracellular vesicles obtained is increased, the vesicle quality is high, and the vesicle particle size is increased. The present invention does not involve animal-derived reagents, is inexpensive, and the extracellular vesicles of EPCs cells amplified have a large number, high purity, and good skin wound repair effect. At the same time, the extracellular vesicles in the present invention have the advantages of being easy to scale up production, convenient for storage and control of batch quality, simple methods for obtaining and using, good damage repair effect, and no immune rejection reaction, and are expected to be prepared into clinical application extracellular vesicles in large quantities. Description of the drawings

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for description in the embodiments. Among them:

[0019] Figure 1 To optimize the composition of the animal-free EPCs culture medium and the cell growth state.

[0020] Figure 2 Results of western blot detection of vesicle positive protein markers (TSG101, Alix, and CD9).

[0021] Figure 3 Results of vesicle number detection.

[0022] Figure 4 Results of using the vesicles prepared from the endothelial progenitor cells derived from the placenta tissue of the present invention for treating skin wound repair. Detailed implementation manners

[0023] In order to make the above objects, features, and advantages of the present invention more obvious and understandable, the following will make a detailed description of the specific implementation manners of the present invention in combination with specific embodiments.

[0024] Example 1:

[0025] Culture of EPCs cell culture medium and EPCs cells derived from placenta tissue:

[0026] Take 10 g of full-term fetal placenta tissue, cut it into minced meat, add 10 mg of type II collagenase, 10 ml of EBM-2 basal medium, and 10 ml of trypsin, and digest in a 37 °C shaker for 1.5 hours. After filtering with gauze, a cell suspension is obtained, and mononuclear cells are obtained using lymphocyte separation medium. The mononuclear cells are resuspended with the EPCs cell culture medium of the present invention or the control group EPCs cell culture medium respectively, and planted in a culture dish pre-coated with human plasma fibronectin for cell induction and amplification.

[0027] The formula of the EPCs cell culture medium of the present invention (Method B): Add 5% (v / v) of serum replacement (human platelet lysate), 100 ng / mL of VEGF, 30 ng / mL of IGF, 6.5 mM of L-glutamine, 0.5 mM of sodium pyruvate, 100 ng / mL of hydrocortisone, and 100 μM of ascorbic acid to the EBM-2 basal medium.

[0028] The formula of the EPCs cell culture medium for the control group (Method A): Add 5% (v / v) of serum replacement (human platelet lysate), 100 ng / mL of VEGF, 30 ng / mL of IGF, 10 ng / mL of bFGF, 100 ng / mL of hydrocortisone, and 100 μM of ascorbic acid to the EBM-2 basal medium.

[0029] Sources of experimental materials: EBM-2 basal medium: LONZA, cc-3156; human platelet lysate: Helios-HPCPLCRL05; VEGF kx-GMP-043, IGF kx-GMP-034, bFGF kx-GMP-007, Beijing Kexin Biotech; hydrocortisone: MCE, HY-N058; ascorbic acid: MCE, HY-B0166.

[0030] The formula of the EPCs cell culture medium of the present invention (Method B) is a further optimization of the formula of the EPCs cell culture medium for the control group (Method A). Figure 1 A). Figure 1 A represents the formula of the EPCs cell culture medium of the present invention (Method B) and the formula of the EPCs cell culture medium for the control group (Method A). Compared with the control culture protocol, the formula of the EPCs cell culture medium of the present invention (Method B) increases L-glutamine and sodium pyruvate and removes the bFGF factor. Under the same culture conditions, the proliferation rate of EPCs cells obtained by culturing with the formula of the EPCs cell culture medium of the present invention (Method B) is significantly higher than that of EPCs cells obtained by culturing with the formula of the EPCs cell culture medium for the control group (Method A). Figure 3 B). Figure 1 B is an optical microscope picture of EPCs cells cultured by the optimized protocol.

[0031] Example 2:

[0032] Identification of EPCs extracellular vesicles:

[0033] The International Society for Extracellular Vesicles (MISEV2018) has stipulated the requirements for vesicle identification, including at least 3 positive proteins (including at least 1 transmembrane protein and 1 cytosolic protein) and 1 negative protein marker. The positive protein markers can be selected from transmembrane proteins CD9, CD63, CD81, and cytosolic proteins TSG101, Alix, HSP70. The negative protein markers can be selected from endoplasmic reticulum protein Calnexin, nuclear protein histone 3, or Golgi protein GM130. The extracellular vesicles obtained by culturing in this system were detected by western blot method and met the at least 3 positive and 1 negative standard stipulated by the International Society for Extracellular Vesicles. Figure 2 A shows the western blot detection results of negative protein markers (Calnexin, GM130) of vesicles obtained by culturing EPCs cells derived from placental tissue with the EPCs cell culture medium formula (Method B) in Example 1. Figure 2 B shows the western blot detection results of positive protein markers (TSG101, Alix, and CD9) of vesicles obtained by culturing EPCs cells derived from placental tissue with the EPCs cell culture medium formula (Method B) in Example 1.

[0034] Example 3:

[0035] The extracellular vesicles obtained by this protocol have significant quantity and quality advantages:

[0036] Seed 1×10 5 cells in a culture dish and culture EPCs cells using two culture media: the EPCs cell culture medium formula (Method B) of the present invention and the control EPCs cell culture medium formula (Method A). The culture conditions are: 37 °C, 5% CO 2 incubator. When the density of primary EPCs cells cultured by one of the protocols reaches 90 - 95% (Method B grows full first), digest the EPCs cells cultured with Method A and Method B media respectively with 1 ml of trypsin, place them in a 37 °C cell incubator for digestion for 2 min, and use 20 ml of PBS to terminate the digestion process. Collect the cell suspension, centrifuge at 1500 rpm for 5 min to obtain cell pellets. Resuspend with 2 ml of medium and count using a cell counting chamber ( Figure 3B). Then the cells were passaged and seeded in a new culture dish. When the cell confluence reached 90 - 95%, the above operations were repeated to obtain cells of different passages. Flow cytometry antibodies were used for immunophenotypic identification to detect the purity of EPC cells in each generation. After the cell density of one of the culture methods reached 90 - 95%, the cell supernatants corresponding to the two culture methods of Method A and Method B in each passage were collected, and extracellular vesicles were enriched by differential centrifugation and ultracentrifugation. The specific centrifugation steps were as follows: (1) The collected supernatant was centrifuged at 300 g for 10 min; (2) The supernatant was taken and centrifuged at 2000 g for 10 min; (3) The supernatant was taken and centrifuged at 10000 g for 30 min; (4) The supernatant was taken and centrifuged at 100000 g for 80 min; (5) The supernatant was discarded, and according to the need, it was resuspended with an appropriate volume of PBS or normal saline to obtain purified extracellular vesicles, and the vesicle size and concentration were detected using an NTA particle size analyzer. In this system, for the EPCs cells prepared from 10 g of placental tissue, the average vesicle size was approximately 143.8 nm, which was better than that of the control culture method (Method A, with an average vesicle size of approximately 127.3 nm)( Figure 3 A, Figure 3 D). The EPCs cell culture medium formula (Method B) of the present invention is an improvement over the control culture method. By detecting the number of extracellular vesicles obtained in each passage, it can be concluded that under the same culture conditions and culture time, the number of vesicles obtained in each passage of the present invention is significantly increased( Figure 3 C). Figure 3 Results of vesicle number and size detection.

[0037] Figure 3 Among them, P1 represents the EPCs cells obtained after the EPCs cells cultured in the present invention are passaged once, and so on. P2 represents the EPCs cells obtained after the EPCs cells cultured in the present invention are passaged twice, and P3 represents the EPCs cells obtained after the EPCs cells cultured in the present invention are passaged three times.

[0038] Example 4:

[0039] Vesicle repair function detection:

[0040] The extracellular vesicles of EPCs cells identified above were used for tissue injury repair function testing. The mice were anesthetized by intraperitoneal injection, placed on their backs, and their four limbs were fixed. The hair on the back was shaved and prepared for skin. A filter paper with a diameter of 1 cm immersed in gentian violet solution was clamped with forceps and pasted on the shaved area, and the paper was gently pressed to make a marked area on the back that was the same size and shape as the paper. The epidermal layer of the marked area was cut off along the marked edge line with scissors. Ten 8The vesicles (50 μl) or an equal volume of PBS as a control were applied to the wound. After 1 minute, 3M tissue glue was sprayed onto the wound, and a protective film was formed after 30 seconds to fix the vesicles at the wound site. The mice were housed separately, the bedding was changed frequently to keep the environment dry and clean, and sufficient feed and water were provided. The tissue damage repair was observed and photographed on days 0, 4, and 8 after treatment. The results showed that the wound repair speed was significantly faster in the group treated with vesicles than in the control group. Figure 4 This is the result of using the vesicles prepared from the endothelial progenitor cells derived from the placenta tissue of the present invention for the treatment of skin wound repair. The present invention first discovers that the extracellular vesicles prepared from the endothelial progenitor cells derived from the placenta have the effect of skin wound repair.

[0041] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

Claims

1. A human endothelial progenitor cell culture medium, characterized in that: The formula of the human endothelial progenitor cell culture medium is: adding 3-10% volume concentration of human platelet lysate, 60-200 ng / mL of VEGF, 20-100 ng / mL of IGF, 2-10 mM of L-glutamine, 0.02-0.75 mM of sodium pyruvate, 50-250 ng / mL of glucocorticoid, and 50-200 μM of reducing agent into the cell basal culture medium; the human endothelial progenitor cells are endothelial progenitor cells derived from human placental tissue.

2. The human endothelial progenitor cell culture medium according to claim 1, characterized in that: The glucocorticoids include one or more of hydrocortisone, cortisone acetate, and prednisone; the reducing agent includes one or more of ascorbic acid, cysteine, and sodium sulfite.

3. The human endothelial progenitor cell culture medium according to claim 1 or 2, characterized in that: The formula of the human endothelial progenitor cell culture medium is: add 5% volume of human platelet lysate, 100 ng / mL VEGF, 30 ng / mL IGF, 6.5 mM L-glutamine, 0.5 mM sodium pyruvate, 100 ng / mL hydrocortisone, and 100 μM ascorbic acid into the basic cell culture medium.

4. The use of the culture medium of human endothelial progenitor cells according to claim 1 in preparing extracellular vesicles, characterized in that: The human endothelial progenitor cells are endothelial progenitor cells derived from human placental tissue, and the extracellular vesicles of the endothelial progenitor cells derived from human placental tissue can be used to prepare skin wound repair drugs.

5. The use according to claim 4, characterized in that: The method for preparing the extracellular vesicles of human placenta tissue-derived endothelial progenitor cells comprises differential centrifugation.

6. The use according to claim 5, characterized in that: The method for preparing the extracellular vesicles of human placental tissue-derived endothelial progenitor cells is as follows: culturing placental tissue-derived endothelial progenitor cells with the human endothelial progenitor cell culture medium, collecting the culture medium supernatant, centrifuging at 300g for 10min; taking the supernatant and centrifuging at 2000g for 10min; taking the supernatant and centrifuging at 10000g for 30min; taking the supernatant and centrifuging at 100000g for 80min; discarding the supernatant to obtain the extracellular vesicles.

7. The use according to any one of claims 4 to 6, characterized in that: The human placenta tissue-derived endothelial progenitor cells are prepared by digesting the placenta tissue with trypsin and collagenase, separating the mononuclear cells using lymphocyte separation fluid, and inducing the primary endothelial progenitor cells with the culture medium.

8. The use according to claim 7, characterized in that: The method also includes passaging the endothelial progenitor cells induced from the human placental tissue to obtain the passaged endothelial progenitor cells.

9. The use according to claim 8, characterized in that: The passaging includes replacing the cell culture medium every 48 to 72 hours.

10. The use according to claim 9, characterized in that: The endothelial progenitor cells are cultured for 2 to 3 days before passage.

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