CN110241081B

CN110241081B - Treg cell amplification medium and in-vitro amplification method

Info

Publication number: CN110241081B
Application number: CN201910506866.2A
Authority: CN
Inventors: 曾宪卓; 党智笙; 汪月
Original assignee: Shenzhen Istem Regenerative Medicine Sci Tech Co ltd
Current assignee: Shenzhen Istem Regenerative Medicine Sci Tech Co ltd
Priority date: 2019-06-12
Filing date: 2019-06-12
Publication date: 2021-06-11
Anticipated expiration: 2039-06-12
Also published as: CN110241081A

Abstract

In order to solve the problems of low proportion of Treg cells and low expansion efficiency of the existing Treg cell culture method, the invention provides an expansion culture medium of Treg cells, and CD3 and/or CD28 antibody and progestogen are added into the expansion culture medium. The invention also provides an in vitro expansion method of the Treg cells, which comprises the following operation steps: obtaining a blood sample containing Treg cells, and separating mononuclear cells from the blood sample; the mononuclear cells are further cultured by a medium to which CD3 and/or CD28 antibodies and a progestin are added. The method for expanding the Treg cells in vitro greatly improves the efficiency of expanding the Treg cells in vitro under the simulated microenvironment of pregnancy.

Description

Treg cell amplification medium and in-vitro amplification method

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an expansion medium and an in-vitro expansion method for Treg cells.

Background

Treg cells (regulatory T cells), otherwise known as suppressor T cells, are a subset of T cells for which the immune regulatory system maintains tolerance to self-antigens and prevents autoimmune diseases. Treg cells are immunosuppressive and either long-term suppress or down-regulate the induction and proliferation of effector T cells.

The surface markers of Treg cells are CD4, CD25 and Foxp3, and since effector T cells also express CD4 and CD25, Treg cells are poorly resolved from CD4+ effector cells. Recent studies have found that the cytokine TGF- β is critical for Treg differentiation from naive CD4+ cells and is very important for maintaining Treg homeostasis.

There are several studies that have shown that modulation of Treg cells can treat autoimmune diseases and cancer, and is beneficial for organ transplantation and wound healing. Clinically, when the Treg cells are used for treating diseases, at least 1X 109 cells need to be infused in a single time, and the Treg cells in peripheral blood only account for 1-2% of mononuclear cells and can meet the clinical requirement only by expanding and culturing at least 50-100 times.

The conventional means for culturing Treg cells is to activate and stimulate mononuclear cells separated from peripheral blood by using CD3+/CD28+ magnetic beads or antibodies, and add IL-2 and TGF-beta into a culture medium. However, because the proportion of Treg cells in peripheral blood is low, the proportion of Treg cells obtained by the traditional amplification method is low, and the traditional amplification method has low efficiency, so that the number of cells meeting clinical use is difficult to obtain.

Disclosure of Invention

Aiming at the problems of low proportion of Treg cells and low amplification efficiency of the existing Treg cell culture method, the invention provides an in-vitro amplification method and an amplification culture medium of Treg cells.

The technical scheme adopted by the invention for solving the technical problems is as follows:

in one aspect, the embodiments of the present invention provide an expansion medium for Treg cells, wherein CD3 and/or CD28 antibodies and progestins are added to the expansion medium.

Optionally, the main body of the amplification medium is AIM-V medium containing 5% autologous serum.

Optionally, the addition amount of the CD3 and/or CD28 antibody is 0.5-10 mug/mL, and the addition amount of the progestogen is 0.5-10 mug/mL;

the progestogen is selected from the group consisting of progesterone.

Optionally, an estrogen is further added into the amplification culture medium, the estrogen is selected from 17 beta-estradiol, and the addition amount of the estrogen is 0.1-10 ng/mL.

Optionally, rIL-2 and TGF-beta are further added into the amplification medium, the addition amount of the rIL-2 is 0.1-300 mu g/mL, and the addition amount of the TGF-beta is 0.1-40 ng/mL.

Optionally, one or more of rIL-1 beta, rIL-4, rIL-5, rIL-6, rIL-7, rIL-8, rIL-9, rIL-10, rIL-17 and rIL-22 are further added into the amplification medium, the addition amount of the rIL-1 beta is 0.1-1 ng/mL, the addition amount of the rIL-4 is 0.01-0.5 ng/mL, the addition amount of the rIL-5 is 1-100 ng/mL, the addition amount of the rIL-6 is 1-100 ng/mL, the addition amount of the rIL-7 is 1-100 pg/mL, the addition amount of the rIL-8 is 1-100 ng/mL, the addition amount of the rIL-9 is 1-100 ng/mL, and the addition amount of the rIL-10 is 1-100 g/mL, the addition amount of the rIL-17 is 1-100 pg/mL, and the addition amount of the rIL-22 is 0.5-1 ng/mL.

In another aspect, the present invention provides a method for expanding Treg cells in vitro, comprising the following steps:

obtaining a blood sample containing Treg cells, and separating mononuclear cells from the blood sample;

the mononuclear cells were further cultured using the amplification medium as described above.

Optionally, the blood sample is peripheral blood or umbilical cord blood;

the "isolation of mononuclear cells from a blood sample" comprises the following operations:

centrifuging the blood sample to obtain lower layer blood cells;

adding PBS with the same volume into blood cells, uniformly mixing to obtain diluted blood, adding TBD separation solution into a centrifuge tube, adding diluted blood with the same volume onto the TBD separation solution, and centrifuging at room temperature;

after centrifugation, sucking and discarding the liquid of the uppermost plasma layer to a position 5mm away from the tunica albuginea layer, and carefully sucking all mixed liquid above the erythrocyte layer into a new centrifugal tube;

adding RPMI1640 into the obtained mixed solution, uniformly mixing, centrifuging, and removing supernatant to obtain cell precipitate;

repeating the operation for multiple times to obtain the separated mononuclear cells.

Alternatively, after "isolating mononuclear cells from a blood sample",

resuspending mononuclear cells in 5% autologous serum AIM-V medium and counting; and controlling the cell concentration at 5X 10⁵Adding the mixture into a culture bottle of AIM-V culture medium containing 5% of autologous serum for culture;

and after culturing for 3-5 h, sucking the culture medium in the culture bottle, then gently washing the bottom surface of the culture bottle by using an AIM-V culture medium containing 5% of autologous serum, transferring the culture bottle to a new culture bottle, and adding an amplification culture medium formed by CD3 and/or CD28 antibody, estrogen and progestogen into the new culture bottle for culturing.

Optionally, the medium and additives in the medium are supplemented during the process of culturing the mononuclear cells, so that the concentration of the additives is kept constant.

During the gestational phase of a mammal, the microenvironment at the maternal-fetal interface facilitates the establishment of pregnancy, maintenance of fertilized egg development and fetal growth. While uncontrolled immune regulation at the maternal-fetal interface can lead to pregnancy failure. The Treg cells of CD4+/CD25+ exist in various stages of pregnancy, inhibit the homogeneous immunological rejection of a mother to a fetus, and the reduction of the function of the Treg cells breaks the maternal-fetal tolerance state and leads to pregnancy failure. Based on the above inspiration, the inventor creatively adds progestogen in the culture medium, aiming at simulating the microenvironment of pregnancy, and unexpectedly finds that the efficiency of expanding Treg cells in vitro is greatly improved under the simulated microenvironment of pregnancy.

Through laboratory tests and statistics, the Treg cells prepared by the method can proliferate by more than 300 times, wherein the cells of CD4+/CD25+/Foxp3+ account for more than 80%. Compared with a culture system only using the traditional IL-2 and TGF-beta, the proliferation capacity of the Treg cells is improved by 5 times, and the purity is improved by 2.3 times. The obtained Treg cells can be directly used for clinical treatment without pre-purification, so that the preparation period is shortened, the production efficiency is improved, and the cost is reduced.

Detailed Description

In order to make the technical problems, technical solutions and advantageous effects solved by the present invention more apparent, the present invention is further described in detail below with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The embodiment of the invention provides an expansion culture medium for Treg cells, wherein CD3 and/or CD28 antibodies and progestagens are added into the expansion culture medium.

The inventor creatively adds the progestogen into the culture medium, aims to simulate a pregnancy microenvironment, and unexpectedly finds that the efficiency of expanding the Treg cells in vitro is greatly improved under the simulated pregnancy microenvironment.

The CD3 and/or CD28 antibodies are used to stimulate T cell activation.

In some embodiments, the body of amplification medium is AIM-V medium containing 5% autologous serum.

In some embodiments, the CD3 and/or CD28 antibody is added in an amount of 0.5-10 μ g/mL and the progestin is added in an amount of 0.5-10 μ g/mL.

The inventors found through a large number of experiments that when the addition amount of the progestogen is within the above range, the effect of inducing Treg cell differentiation and proliferation is good, and when the addition amount of the progestogen is low, the effect of promoting proliferation is relatively reduced; when the addition amount of the progestogen is relatively high, the effect of promoting proliferation is similarly improved with respect to the addition amount within the above range.

In some embodiments, the CD3 and/or CD28 antibody is selected from the group consisting of CD3/CD28 monoclonal antibodies.

In some embodiments, the progestogen is selected from one or more of a natural progestogen, a synthetic progestogen, and synthetic derivatives thereof.

In one embodiment, the progestogen is selected from the group consisting of progesterone (progasterone).

In other embodiments, the progestogen may also be selected from derivatives of progesterone (progasterone).

In some embodiments, due to the fact that the estrogen level is increased during pregnancy, in order to better simulate a pregnancy microenvironment, the culture medium is also added with estrogen, and the addition amount of the estrogen is 0.1-10 ng/mL.

In one embodiment, the estrogen is selected from 17 β -estradiol.

In some embodiments, the medium is further added with rIL-2 (recombinant interleukin-2) and TGF-beta (transforming growth factor-beta), wherein the addition amount of the rIL-2 is 0.1-300 mu g/mL, and the addition amount of the TGF-beta is 0.1-40 ng/mL.

The rIL-2 and the TGF-beta have the effect of promoting the proliferation of T cells and can improve the yield of Treg cells.

In some embodiments, the medium is further supplemented with one or more of rIL-1 β, rIL-4, rIL-5, rIL-6, rIL-7, rIL-8, rIL-9, rIL-10, rIL-17, and rIL-22.

By adding the multiple recombinant interleukins, the cytokine environment in a microenvironment of pregnancy can be better simulated, so that the differentiation of the naive CD4+ cells into Treg cells is better promoted, and the proliferation of the Treg cells is promoted.

In some embodiments, the amount of the rIL-1 beta is 0.1-1 ng/mL, the amount of the rIL-4 is 0.01-0.5 ng/mL, the amount of the rIL-5 is 1-100 ng/mL, the amount of the rIL-6 is 1-100 ng/mL, the amount of the rIL-7 is 1-100 pg/mL, the amount of the rIL-8 is 1-100 ng/mL, the amount of the rIL-9 is 1-100 ng/mL, the amount of the rIL-10 is 1-100 pg/mL, the amount of the rIL-17 is 1-100 pg/mL, and the amount of the rIL-22 is 0.5-1 ng/mL.

Another embodiment of the present invention provides an in vitro expansion method of Treg cells, comprising the following steps:

the mononuclear cells are further cultured by a medium to which CD3 and/or CD28 antibodies and a progestin are added.

In some embodiments, the blood sample is peripheral blood or umbilical cord blood.

The peripheral blood and the umbilical cord blood both contain a certain amount of T cells, and can be used for inducing differentiation and proliferation into Treg cells.

In some embodiments, the "isolation of mononuclear cells from a blood sample" comprises the following operations:

centrifuging the blood sample to obtain lower layer blood cells;

adding Phosphate Buffer Saline (PBS) with the same volume into blood cells, uniformly mixing to obtain diluted blood, adding TBD separation liquid into a centrifuge tube, adding diluted blood with the same volume into TBD separation liquid (human peripheral blood lymphocyte separation liquid), and centrifuging at room temperature;

The PBS may also be replaced with saline.

By way of example, in one embodiment, the blood sample is centrifuged at 3000rpm for 10min at 4 ℃ to separate the lower layer of blood cells;

adding PBS with the same volume into blood cells, uniformly mixing to obtain diluted blood, selecting a 50ml centrifuge tube, adding 15ml TBD separating medium into the centrifuge tube, placing the centrifuge tube at room temperature (18-22 ℃), adding diluted blood with the same volume into the TBD separating medium, and centrifuging at room temperature, wherein the yield is reduced by centrifuging at low temperature, the cells are easily inactivated by centrifuging at high temperature, and the cells are slowly increased and decreased by using 1-step acceleration.

After centrifugation, the uppermost plasma layer liquid was aspirated down to 5mm from the tunica albuginea layer with a pipette, and then all mixed liquid above the erythrocyte layer was carefully aspirated into a new centrifuge tube.

RPMI1640 (Roswell Park mechanical Institute 1640) was added to the mixture to make up to 40ml, and the mixture was mixed, centrifuged at 400g for 10min, and the supernatant was discarded to obtain a cell precipitate.

Then 10ml RPMI1640 was added to the cell pellet, mixed well and centrifuged at 300g for 10min, the supernatant was discarded, and the procedure was repeated twice.

Isolated mononuclear cells were obtained.

In some embodiments, after "isolating mononuclear cells from a blood sample",

the autologous serum refers to the serum of the blood sample donor, specifically, the autologous serum of the embodiment is obtained from the plasma on the upper layer separated in the operation of "obtaining the blood cells on the lower layer by centrifugal separation of the blood sample", and the autologous serum can be obtained by placing the plasma in a 56 ℃ water bath for heat inactivation for 30min, so that the blood sample can be fully utilized, and multiple blood drawing is avoided.

The components of the amplification medium are the same as those of the amplification medium, and thus are not described in detail.

In some embodiments, the medium and additives in the medium are supplemented during the culturing of the mononuclear cells such that the concentration of the additives remains constant.

Specifically, fresh medium containing autologous serum and IL-2 was supplemented every 3 days of culture, and TGF- β, rIL-1 β, rIL-4, rIL-5, rIL-6, rIL-7, rIL-8, rIL-9, rIL-10, rIL-17, rIL-22, progesterone (progesterone) and 17 β -estradiol were added, with the concentrations being maintained constant.

Culturing to day 12, collecting cells, and detecting the number of cells and the proportion of cells of CD4+ CD25+ Foxp3 +.

The present invention will be further illustrated by the following examples.

Example 1

This example illustrates the method for the ex vivo expansion of Treg cells disclosed in the present invention, comprising the following operating steps:

isolation of peripheral blood mononuclear cells: collecting 50mL of peripheral blood of healthy adult, centrifuging at 4 ℃ and 3000rpm for 10min, sucking out the upper layer of plasma, placing in a 56 ℃ water bath pot for heat inactivation for 30min, and placing in a 4 ℃ refrigerator for later use. The lower layer of blood cells was diluted with an equal volume of physiological saline and an equal volume of TBD cell separation medium was slowly added. The cells were centrifuged at 700g for 25min at 22 ℃ to obtain mononuclear cells. By counting and flow-through detection, about 7.2X 10 is obtained⁷And single nuclear cells, wherein the Treg cells of CD4+/CD25+ account for 1.9 percent.

And (3) culturing the Treg cells: the obtained mononuclear cells were adjusted to a concentration of 5X 10 by using a heat-inactivated X-VIVO medium containing 5% autologous serum⁵mL, transferred to a T125 cell culture flask and incubated in a cell incubator for 4 h. The culture medium containing the suspended cells was then aspirated, leaving about 5ml, and the bottom of the flask was gently flushed to float the settled suspended cells. Transfer of cells to a New T125 flaskTo this, CD3/CD28 monoclonal antibody (2. mu.g/mL) and rIL-2 (100. mu.g/mL), TGF-. beta.4 (4 ng/mL), rIL-1. beta.200 pg/mL), rIL-4 (90 pg/mL), rIL-5 (10 ng/mL), rIL-6 (10 ng/mL), rIL-7 (15 pg/mL), rIL-8 (17.6 ng/mL), rIL-9 (10 ng/mL), rIL-10 (20 pg/mL), rIL-17 (20 g/mL), rIL-22 (0.7 ng/mL), progesterone (3. mu.g/mL) and 17. beta. -plastadiol (1 ng/mL) were added. Controlling the cell concentration not to exceed 1 × 10⁶Each 3 days, half the volume of fresh X-VIVO medium containing 5% autologous serum and rIL-2, TGF- β, rIL-1 β, rIL-4, rIL-5, rIL-6, rIL-7, rIL-8, rIL-9, rIL-10, rIL-17, rIL-22, progesterone and 17 β -estradiol was added, with the additive concentration being maintained.

Culturing to day 12, detecting the total cell number to be 5.4 × 10⁸And the Treg cells with amplification fold 397 times and CD4+/CD25+/Foxp3+ account for 92.6 percent.

Example 2

This example is intended to illustrate the method for the in vitro expansion of Treg cells disclosed in the present invention, comprising most of the operating steps of example 1, with the following differences:

progesterone was added in an amount of 0.5. mu.g/mL.

Culturing to day 12, detecting the total cell number to be 4.7 × 10⁸And the amplification factor is 345 times, and the Treg cells of CD4+/CD25+/Foxp3+ account for 85.2 percent.

Example 3

progesterone was added in an amount of 10. mu.g/mL.

Culturing to day 12, detecting the total cell number to be 5.8 × 10⁸And the Treg cells with the amplification factor of 426 times and the expansion ratio of CD4+/CD25+/Foxp3+ account for 63.2 percent.

Example 4

in the culture medium without adding rIL-1 beta, rIL-4, rIL-5, rIL-6, rIL-7, rIL-8, rIL-9, rIL-10, rIL-17 and rIL-22.

Culturing to day 12, detecting total cell number of 2.8 × 10⁸And the expansion ratio is 205 times, and the Treg cells of CD4+/CD25+/Foxp3+ account for 85.3 percent.

Example 5

no 17 β -estradiol was added to the medium.

Culturing to day 12, detecting total cell number of 4.6 × 10⁸And the Treg cells with the amplification factor of 352 and the CD4+/CD25+/Foxp3+ account for 84.9 percent.

Comparative example 1

This comparative example is intended to illustrate by comparison the method for the in vitro expansion of Treg cells disclosed in the present invention, comprising most of the operating steps of example 1, with the following differences:

progesterone was not added to the medium.

Culturing to day 12, detecting total cell number of 4.1 × 10⁸And the Treg cells with the amplification factor of 301 are 84.7 percent of the Treg cells with the amplification factor of CD4+/CD25+/Foxp3 +.

The detection results of the embodiment 1-5 and the comparative example 1 are compared, so that the in-vitro expansion method for the Treg cells simulating the pregnancy microenvironment, provided by the invention, can effectively improve the efficiency of in-vitro expansion of the Treg cells, ensure the ratio of the Treg cells in the final culture product and obtain the Treg cells with higher purity.

From the test results of example 2 and comparative example 1, it is understood that when the concentration of progasterone is too high, the proportion of positive cells (Treg cells) is reduced, but the expansion rate is still improved, which is advantageous for promoting the expansion of cells.

Comparing the detection results of example 1, example 4 and example 5, it can be seen that the pregnancy microenvironment can be better simulated and the expansion efficiency of Treg cells can be further improved by adding recombinant interleukins such as rIL-1 beta, rIL-4, rIL-5, rIL-6, rIL-7, rIL-8, rIL-9, rIL-10, rIL-17, rIL-22 and estrogens such as 17 beta-estradiol on the basis of the present invention.

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 and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims

1. An expansion culture medium for Treg cells, which is characterized in that a CD3 and/or CD28 antibody and a progestogen are added into the expansion culture medium, wherein the addition amount of the progestogen is 0.5-3 mu g/mL;

the amplification culture medium is also added with estrogen, and the estrogen is selected from 17 beta-estradiol.

2. The expansion medium for Treg cells according to claim 1, wherein the bulk of the expansion medium is AIM-V medium containing 5% autologous serum.

3. The Treg cell expansion medium of claim 1, wherein the CD3 and/or CD28 antibody is added in an amount of 0.5-10 μ g/mL;

the progestogen is selected from the group consisting of progesterone.

4. The expansion medium for Treg cells according to claim 1, wherein the estrogen is added in an amount of 0.1-10 ng/mL.

5. The Treg cell expansion medium according to claim 1, wherein rIL-2 and TGF- β are further added to the expansion medium, wherein the rIL-2 is added in an amount of 0.1 to 300 μ g/mL, and the TGF- β is added in an amount of 0.1 to 40 ng/mL.

6. The Treg cell expansion medium according to claim 1, wherein one or more of rIL-1 β, rIL-4, rIL-5, rIL-6, rIL-7, rIL-8, rIL-9, rIL-10, rIL-17 and rIL-22 are added to the expansion medium, the amount of rIL-1 β added is 0.1 to 1 ng/mL, the amount of rIL-4 added is 0.01 to 0.5 ng/mL, the amount of rIL-5 added is 1 to 100 ng/mL, the amount of rIL-6 added is 1 to 100 ng/mL, the amount of rIL-7 added is 1 to 100 pg/mL, the amount of rIL-8 added is 1 to 100 ng/mL, and the amount of rIL-9 added is 1 to 100 ng/mL, the addition amount of the rIL-10 is 1-100 pg/mL, the addition amount of the rIL-17 is 1-100 pg/mL, and the addition amount of the rIL-22 is 0.5-1 ng/mL.

7. An in vitro expansion method of Treg cells, comprising the following operating steps:

the mononuclear cell is further cultured by using the amplification medium according to any one of claims 1 to 6.

8. The method for the in vitro expansion of Treg cells according to claim 7, wherein said blood sample is peripheral blood or umbilical cord blood;

centrifuging the blood sample to obtain lower layer blood cells;

adding PBS with the same volume into blood cells, uniformly mixing to obtain diluted blood, adding lymphocyte separation liquid into a centrifuge tube, adding diluted blood with the same volume onto the lymphocyte separation liquid, and centrifuging at room temperature;

9. The method for the in vitro expansion of Treg cells according to claim 7, wherein, after the "isolation of the mononuclear cells from the blood sample",

AIM-V culture with 5% autologous serumSuspending the mononuclear cells by weight, and counting; and controlling the cell concentration at 5X 10⁵Adding the mixture into a culture bottle of AIM-V culture medium containing 5% of autologous serum for culture;

10. The method for the in vitro expansion of Treg cells according to claim 7, wherein the culture medium and the additives contained in the culture medium are supplemented during the culture of the mononuclear cells, such that the concentration of the additives is maintained constant.