CN114990068B

CN114990068B - Preparation method and application of umbilical cord mesenchymal stem cell exosome

Info

Publication number: CN114990068B
Application number: CN202210526064.XA
Authority: CN
Inventors: 陈新峰; 周凌霄; 张毅; 张震
Original assignee: First Affiliated Hospital of Zhengzhou University
Current assignee: First Affiliated Hospital of Zhengzhou University
Priority date: 2022-05-16
Filing date: 2022-05-16
Publication date: 2023-09-22
Anticipated expiration: 2042-05-16
Also published as: CN114990068A

Abstract

The invention discloses a preparation method and application of umbilical cord mesenchymal stem cell exosomes, wherein the preparation method mainly comprises the following steps: (1) Frozen P3 generation MSCs are taken and cells are recovered, and the cells are 1 multiplied by 10 per dish ⁶ Uniformly spread in 10cm cell culture dishes, and adding 10mL MSCs serum-free medium into each dish; (2) And (2) when the cell fusion rate in the step (1) is 90%, transfecting the synthesized hsa-miR-3194-3p micrometers and NC into MSC, collecting cell culture supernatant for exosome extraction after 48 hours, and collecting supernatant for exosome extraction after 48h hours of cell culture. The use of MSC exosomes expressing miR-3194-3p is able to inhibit cytokine production without affecting CAR-T cell effector function.

Description

Preparation method and application of umbilical cord mesenchymal stem cell exosome

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a preparation method and application of umbilical cord mesenchymal stem cell exosomes.

Background

Chimeric antigen receptor (chimeric antigen receptor, CAR) T cell therapy is one of the most promising therapeutic approaches in the field of tumor therapy at present, and achieves exciting effects in hematological tumor therapy, a variety of autologous CAR-T cell therapy products have also been marketed in bulk. However, this highly effective tumor treatment also has certain limitations in clinical treatment, especially cytokine release syndrome (cytokine release syndrome, CRS), which can be life threatening for some patients. The main means for controlling CRS clinically at present include using IL-6 receptor blocker tolizumab injection or glucocorticoid, but these treatments have a certain limitation, when patients are actively infected, tolizumab injection is used to aggravate infection, and glucocorticoid may affect the function of CAR-T cells, so it is necessary to explore a new strategy for controlling CRS.

Mesenchymal stem cells (mesenchymal stem cell, MSCs) are a cell with immunomodulatory effects. Studies have shown that MSCs can be used to treat COVID-19-induced CRS, and that CAR-T cell therapy-related CRS has a certain similarity to the pathological course of COVID-19-induced CRS. We therefore consider the use of MSCs as one of the means to control CAR-T cell therapy-related CRS. The exosomes are spherical vesicles composed of lipid bilayer membranes and contain complex and abundant active substances such as proteins and nucleic acids. Mesenchymal stem cell exosomes (mesenchymal stem cell-derived exosomes, MSC-Exos) are spherical vesicles secreted by MSCs, contain many anti-inflammatory compounds, and regulate immune responses by interacting with immune effector cells. In the treatment of autoimmune diseases, MSC-Exos have received a great deal of attention as carriers of cell-free therapies, as they not only carry most of the therapeutic effects of MSCs themselves, but also reduce concerns about the safety of injected living cells. MSC-Exos has significant advantages in clinical treatment and may completely replace MSCs in the future. Thus, the present study suggests that MSC-Exos is capable of alleviating CRS caused by CAR-T cell therapy or novel coronavirus infection.

It has been reported in the literature that CRS induced during CAR-T cell therapy is intimately associated with macrophages, and that cytokines that play an important role in the development of CRS are IL-1 β and IL-6, whereas IL-1 β is the "switch" of the overall inflammatory process and is in a position upstream of the overall inflammatory process. However, CRS caused by CAR-T cell killing is mainly related to which inflammatory body is activated, and MSC-Exos is modified to have a stronger CRS inhibiting capability, so no relevant report exists at present.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a preparation method and application of umbilical mesenchymal stem cell exosomes, which can inhibit cytokine production by using umbilical cord MSC-Exos containing miR-3194-3p under the condition of not affecting the effector function of CAR-T cells.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

the invention provides a preparation method of umbilical mesenchymal stem cell exosomes, which mainly comprises the following steps:

(1) Frozen P3 generation MSCs are taken and cells are recovered, and the cells are 1 multiplied by 10 per dish ⁶ Uniformly spread in 10cm cell culture dishes, and adding 10mL MSCs serum-free medium into each dish;

(2) When the cell fusion rate in the step (1) is 90%, removing the MSCs serum-free culture medium, washing the cells once by using PBS, replacing the culture medium with MSC-Exos extraction culture medium, transfecting the synthesized hsa-miR-3194-3p mimics into mesenchymal stem cells, culturing for 48 hours, culturing the cells for 48h, collecting the supernatant, and extracting exosomes by using an ultracentrifugation method.

The preparation method of umbilical cord mesenchymal stem cell exosomes as described above, wherein in the above steps, MSCs culture supernatant is centrifuged at 2000g for 20min to remove large fragments and dead cells, after centrifugation, the supernatant is collected and centrifuged at 10,000×g for 1 hour to remove small cell fragments by an ultracentrifuge, after centrifugation, the supernatant is collected and filtered by a 0.22 μm filter, centrifuged at 100,000×g for 80min, the supernatant is discarded, and after re-suspending precipitation by PBS, the supernatant is centrifuged at 100,000×g for 80min to remove contaminating proteins, and then the precipitated exosomes are re-suspended by 200 μl of PBS, and stored at-80 ℃ for later use.

The application of the umbilical cord mesenchymal stem cell exosome in preparing the medicine for treating cytokine release syndrome.

Cytokine levels were measured as described above.

The application of the umbilical cord mesenchymal stem cell exosome in preparing medicaments for treating novel cytokine release syndromes related to coronavirus infection.

As applied above, the detection process is as follows:

s1, balancing the kit to room temperature, preparing 1.5 mL EP pipes according to the number of samples, adding 25 mu L of detection buffer solution into each EP pipe, and adding 25 mu L of detection sample;

s2, taking out the captured microspheres, carrying out vortex oscillation on a vortex instrument for more than 1 min, diluting the captured microspheres by 13 times by using a detection buffer solution to prepare a captured microsphere mother solution, and adding 25 mu L of diluted captured microspheres into each tube;

s3, incubating for 2 hours at 800 rpm/min on a normal-temperature shaking table in a dark place;

s4, centrifuging for 5 min 250 g after incubation, removing liquid, adding 300 mu L of 1 Xwashing liquid, centrifuging for 5 min 250 g after incubation for 1 min, and discarding liquid;

s5, adding 25 mu L of detection antibody into each hole, and incubating for 1 hour at 800 rpm/min in a shaking table at normal temperature in a dark place; then 25. Mu.L of detection antibody is added, incubated for half an hour at 800 rpm/min in a dark place on a shaking table at normal temperature, centrifuged at 250 g for 5 min, the liquid is discarded, 300. Mu.L of 1 Xwashing liquid is added to each well, and after incubation for 1 min, the liquid is discarded after centrifugation at 250 g for 5 min. mu.L of 1 Xwash solution was added to each well, and the wells were resuspended and then examined by flow cytometry.

Compared with the prior art, the invention can obtain the following technical effects:

1. the main means for clinically controlling CRS comprises using IL-6 receptor blocker tolizumab injection or glucocorticoid, but the treatment means have certain limitations, when a patient is subjected to active infection, the tolizumab injection can aggravate the infection, and the application of the glucocorticoid can influence the function of CAR-T cells.

2. The invention can effectively inhibit CRS caused by killing the CAR-T cells without affecting the function of the CAR-T cells, and compared with the cells per se, the exocrine body has small volume, low immune response degree and easier preservation, thus having better prospect in clinical application than the simple cells.

Drawings

FIG. 1; a: WB detects the expression of exosome-specific proteins CD9 and Tsg 101; b: NTA particle size detection super-isolate diameter range, C and D: observing morphological characteristics of the super-ionized extract by a transmission electron microscope;

FIG. 2; a: a pattern diagram for detecting the influence of hUC-MSC-Exos on the killing function of CAR-T cells by flow cytometry; b: a statistical graph of the streaming results; and (3) injection: n.s. no statistical significance

FIG. 3; detecting the relative expression quantity of hsa-miR-3194-3p in hUC-MSCs and hUC-MSC-Exos by qRT-PCR; (***p<0.001）

FIG. 4; the effect of adding hsa-miR-3194-3 p-expressing hUC-MSC-Exos to a co-incubation system on CRS caused by killing of CAR-T cells is detected by a multi-factor detection method. (*p<0.05, **p<0.01, ****p<0.0001）。

Detailed Description

The following will describe embodiments of the present invention in detail by referring to examples, so that the implementation process of how to apply the technical means to solve the technical problems and achieve the technical effects of the present invention can be fully understood and implemented.

Examples

The invention discloses a preparation method of umbilical cord mesenchymal stem cell exosomes, which mainly comprises the following steps:

(2) When the cell fusion rate in the step (1) is 90%, removing the MSCs serum-free culture medium, washing the cells once by using PBS, replacing the culture medium with MSC-Exos extraction culture medium, transfecting the synthesized hsa-miR-3194-3p mimics into the MSCs, culturing for 48 hours, culturing the cells for 48h, collecting the supernatant, and extracting exosomes by using an ultracentrifugation method.

The application of the umbilical cord mesenchymal stem cell exosome in preparing medicines for treating cytokine release syndrome.

Cytokine levels were measured as described above.

As applied above, the detection process is as follows:

Experimental method

1. Co-incubation experiments with CD19-CAR-T, raji and MDM

The infected CD19-CAR-T cells, raji cells and induced MDM were collected separately, centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and then a proper amount of fresh medium was added, and the cells were counted after resuspension. According to 2.5X10 per well ⁵ CAR-T cells, 2.5X10 ⁵ MDM and 5×10 ⁵ The number of Raji cells was plated in 12-well plates with a final volume of 2 mL per well of medium.

2. CD19-CAR-T cell killing experiments

2.1 CFSE-labeled target cells

Raji cells were first labeled with CFSE. After cells were collected, washed once with PBS, resuspended in 1 mL serum-free medium, then 1 μl CFSE (5 mM) dye was added, mixed well, placed in a cell incubator for half an hour, and vortexed and mixed well once every 5 min. After the incubation, the supernatant was centrifuged off, staining was stopped with medium containing 20% FBS, and the supernatant was centrifuged off. Cells were washed 3 times as above and then resuspended in appropriate amount of RPMI 1640.

2.2 Cell co-incubation

According to 2.5X10 per well ⁵ CAR-T cells, 2.5X10 ⁵ MDM and 5×10 ⁵ The number of Raji cells was plated in 12-well plates for co-incubation and two days later examined by flow cytometry.

2.3 Apoptosis detection

After the cell co-incubation, cell pellet was collected into a 1.5 mL centrifuge tube, washed once with flow-through loading buffer, and the supernatant was discarded by centrifugation. Cells were resuspended in 60. Mu. L Annexin V Binding Buffer, 1. Mu.L of Annexin V flow antibody was added and incubated at 4℃for 15 min in the absence of light. 1 mu L of PI is added before the machine is started, and the machine is started in a flow mode for detection.

2.4 CAR-T cell proliferation assay

Cell proliferation assay assays were performed using eFluor 670 cell proliferation dye. Collecting CD19-CAR-T cells into a centrifuge tube, centrifuging, removing supernatant, adding sterile PBS, and washingCells were washed twice. Cells were resuspended in PBS to twice the final desired cell concentration. 10 mu M Fluor 670 cell proliferation dye equilibrated to room temperature was added to an equal volume of sterile PBS and blown down uniformly. Adding the Fluor 670 bodies into the cell suspension while blowing, and placing the cells in a cell incubator for incubation for 10 min in a dark place. 5mL of pre-chilled 20% serum medium was added and incubated on ice for 10 min.1500 After centrifugation at rpm for 5 min, the supernatant was discarded, 5mL of pre-chilled 20% serum-containing medium was added and incubated on ice for 5 min. The culture medium was washed twice more, the supernatant was discarded, and the culture medium was resuspended in complete medium, and the culture medium was plated in 96-well plates at a cell amount of 5X 104 cells per well, and the exosome amount added per well was 30. Mu.g/mL. Raji cells and MDM were treated according to the treatment method in 1.20, counted and plated in 96-well plates, and 1X 10 Raji cells were added to each well according to the experimental requirements ⁵ MDM was added 5X 10 per well ⁴ . Fluorescence intensities of 670 were measured by flow cytometry on days 0, 1, 2, and 3, respectively.

3. Transfection of MSCs with miRNA

The frozen P3 generation MSCs are taken for resuscitating the cells. 1X 10 cells per dish after resuscitating ⁶ The cells of (2) are spread in a 10cm cell culture dish, added with MSCs serum-free basal medium, placed in a cell culture box for culture, and transfected when the cell fusion degree reaches about 80 percent. The synthesized hsa-miR-3194-3p chemicals and NC powder were centrifuged at 3000 rpm for 5 min, and 125. Mu.L of enzyme-free water was added to make them fully dissolved. 1.5 mL EP tubes were taken, 500. Mu.L of jetPRIME buffer was added to each tube, followed by 25. Mu.L of the miRNA to be transfected. Vortex 10 s on vortex meter thoroughly, then add 20 μl of jetPRIME reagent, vortex 10 s again, leave at room temperature for 15 min, gently add drop wise to MSCs. And (3) placing the cells in a cell culture box for culturing for 4-6 hours, discarding the original culture medium, washing the cells once by using PBS, and changing the cells into a fresh MSC-Exos extraction culture medium. After 48 hours, the cell culture supernatant was harvested for exosome extraction. PBS was added to the cell culture dish to wash the cells once, after the supernatant was discarded, 0.5 mL RNAiso Plus was added, and after thoroughly blowing and mixing, the cells were placed in an enzyme-free 2 mL EP tube and stored in a-80℃refrigerator for subsequent transfection efficiency detection.

4. Exosomes expressing hsa-miR-3194-3p can kill CAR-T cells-induced CRS

Referring to FIGS. 1-4, it was demonstrated that hsa-miR-3194-3p was indeed able to inhibit expression of inflammatory small associated genes, so we added the hsa-miR-3194-3 p-containing exosomes to the co-incubation system, and proved by multi-factor detection experiments, the addition of hsa-miR-3194-3 p-containing exosomes was indeed able to significantly inhibit CAR-T cell killing-induced CRS.

The application of the umbilical cord mesenchymal stem cell exosome in preparing the medicine for treating the novel cytokine release syndrome of coronavirus.

The present invention may be summarized in other specific forms without departing from the spirit or essential characteristics thereof. The above-described embodiments of the invention are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims

1. The application of umbilical cord mesenchymal stem cell exosomes in preparing medicaments for treating cytokine release syndrome is characterized in that the preparation method of the umbilical cord mesenchymal stem cell exosomes mainly comprises the following steps:

(1) Frozen P3 generation MSCs are taken and cells are recovered, and the cells are 1 multiplied by 10 per dish ⁶ Uniformly spread in 10cm cell culture dishes, and adding 10mL of MSCs serum-free culture medium into each dish;

(2) Removing MSCs serum-free medium when the cell fusion rate is 90% in the step (1), washing the cells once by using PBS, transfecting synthesized hsa-miR-3194-3p micrometers into the MSCs, culturing for 48 hours, centrifuging the MSCs culture supernatant for 20 minutes by 2000g, removing large fragments and dead cells, collecting the supernatant after centrifugation, centrifuging for 1 hour by using an ultracentrifuge for removing small cell fragments by using 10,000 x g, collecting the supernatant after centrifugation, filtering the supernatant by using a filter of 0.22 mu m, centrifuging for 80 minutes by using 100,000 x g, discarding the supernatant, re-suspending the precipitate by using PBS, centrifuging for 80 minutes by using 100,000 x g to remove polluted proteins, re-suspending the precipitated exosomes by using 200 mu L of PBS, and storing the exosomes at the temperature of-80 ℃ for later use.

2. The use according to claim 1, wherein: the application of umbilical cord mesenchymal stem cell exosomes in preparing medicaments for treating cytokine release syndromes caused by novel coronavirus infection.