CN114480279A

CN114480279A - Efficient separation culture technology for human blood immune cells CD4T

Info

Publication number: CN114480279A
Application number: CN202210053066.1A
Authority: CN
Inventors: 王臣
Original assignee: Jilin Zhencheng Pharmaceutical Co ltd
Current assignee: Jilin Zhencheng Pharmaceutical Co ltd
Priority date: 2022-01-18
Filing date: 2022-01-18
Publication date: 2022-05-13

Abstract

The invention discloses a high-efficiency separation culture technology of human blood immune cells CD4T, the separation culture of CD4T comprises two parts, namely a separation method of mononuclear cells immune cells in human blood and an in-vitro culture amplification method of mononuclear cells in human blood.

Description

Efficient separation culture technology for human blood immune cells CD4T

Technical Field

The invention relates to a culture technology, in particular to a high-efficiency separation culture technology of human blood immune cells CD4T, belonging to the technical field of separation culture of biological cells in vitro immune cells.

Background

Immune cells (immuno cells) are commonly called as leucocytes, and comprise lymphocytes, various phagocytes and the like, and also particularly refer to lymphocytes which can recognize antigens and generate specific immune responses. Lymphocytes are the basic components of the immune system and widely distributed in vivo, and mainly T lymphocytes and B lymphocytes are activated (activated) by antigen stimulation, divide, proliferate and generate specific immune response. In addition to T lymphocytes and B lymphocytes, there are also K lymphocytes and NK lymphocytes, of four types. T lymphocytes are a versatile cell population. Besides lymphocytes, the cells involved in the immune response include plasma cells, granulocytes, mast cells, antigen presenting cells and cells of the mononuclear phagocyte system, which are mainly in our blood, and when a problem occurs in that organ of our body, the blood sends the immune cells to the problem part for fighting, thereby eliminating bacteria and viruses entering the body and protecting the cells and organs in our body. When the immune cell population in our body is lack, the body can not timely eliminate the exogenous invading bacteria and viruses and the self-variant cells, and then the body can generate a total disease, such as Hepatitis B (HBV) patients and cancers, which are directly related to the immune cells.

The immune cell population is efficiently separated from the periphery, cultured and amplified in vitro, and clinically applied or frozen after being amplified to a certain number, and the process has one technical difficulty, and the technical difficulty is mainly that enough immune cell seeds in peripheral blood are firstly obtained and cultured and amplified in vitro. The procedure is now to extract 50ml of peripheral blood from a patient, to isolate T lymphocytes from the blood using a T lymphocyte isolate, and to perform subsequent in vitro culture expansion after the isolation. The amount of the culture reagent used for the length of the culture period is directly related to the amount of the culture reagent obtained by the seed cells. The invention greatly improves the number of the primary seed cells of more immune cell groups harvested in peripheral blood, changes the culture method, shortens the culture period, and greatly improves the activity and the number of the in vitro immune cells.

Disclosure of Invention

The invention aims to provide a high-efficiency separation and culture technology of human blood immune cells CD4T, so as to solve the problems in the background technology.

In order to achieve the purpose, the invention provides the following technical scheme: a high-efficiency separation culture technology of human blood immune cells CD4T is disclosed, wherein the separation culture of CD4T comprises two parts of a separation method of mononuclear cell immune cells in human blood and an in vitro culture and amplification method of mononuclear cells in human blood, and the specific contents are as follows:

firstly, the method comprises the following steps: the instrument for preparing the separated blood comprises three parts, namely a centrifuge tube, an anticoagulant, a separation gel, and an immunocyte separation gel, wherein the immunocyte separation gel is a hydrophobic organic compound, and the action principle is as follows: typically, the specific gravity of serum is about 1.02, and the specific gravity of red blood cells is: 093, polymorphonuclear leukocyte specific gravity: 1.092, platelet specific gravity is about: 1.032, the specific gravity of the mononuclear cells is as follows: 1.076-1.090, maintaining the specific gravity of the separation gel at 1.77, forming an isolation layer among serum, white blood cells and red blood cells by the immune cell separation gel under the action of centrifugal force, placing 5ml of the separation gel at the bottom of a 50ml centrifuge tube, sequentially adding polyolefin, polyester and propylene into the centrifuge tube, centrifuging for 3500r/min, centrifuging for 15min, and preparing the centrifuge tube with the immune cell separation gel for later use;

secondly, the method comprises the following steps: extracting 2.0ml heparin anticoagulant by using a 50ml syringe, pushing and pulling the syringe several times to wet the wall of the syringe by heparin, replacing a new syringe needle, extracting 40ml peripheral venous autoblood, placing 40ml anticoagulant whole blood into a centrifuge tube of 50ml separation gel, balancing the centrifuge tube, 3500r/min, centrifuging for 15 minutes to obtain the separation gel, namely forming an isolation layer among serum, white blood cells and red blood cells, keeping the serum in the original state, keeping the serum unchanged, tightly adhering the isolation layer to the wall of the test tube, avoiding the influence of fibrin and hemolysis, adhering a white membrane layer on the separation gel, wherein the white membrane layer is a target cell layer;

thirdly, the method comprises the following steps: taking out the centrifuge tube, discarding the supernatant to the middle layer, reserving 15ml of plasma, gently and repeatedly blowing and beating the plasma to the surface of the immune cell separation gel by using a suction tube, completely blowing the white membrane on the separation gel into the plasma, putting the blown and beaten plasma into a new centrifuge tube, cleaning for 1-2 times, transferring the separated cells into a 50ml centrifuge tube, adding 30ml of saline, blowing and beating by using the suction tube, balancing by using a centrifuge after blowing and beating, and centrifuging in the centrifuge for 2200r/min and rotating for 20 min;

fourthly: the immune cell culture solution is prepared by adding 8% fetal calf serum into 1000ml of immune cell culture medium in bottle A, adding 200 ten thousand U of interleukin 2, adding 10mg of thymopentin, adding 500 ten thousand U of interferon a2B1m, and adding immune cell culture solution in bottle B after 12 days of culture: adding 8% fetal calf serum into 1000ml of immune cell culture medium, adding 200 million U of interleukin 2 in two bottles, adding 10mg of thymopentin, adding 500 million U of interferon a2b1m, and adding 0.25g of BCG;

fifth, the method comprises the following steps: taking out the centrifuge tube, removing supernatant, leaving precipitate, adding 20ml of immune cell culture solution in an A bottle into the precipitated tissue, repeatedly blowing and beating the precipitate by using a bus dropper to completely blow off the precipitate, transplanting the precipitate into a T82 cell culture bottle after blowing, putting a T82 cell culture bottle into an incubator of 5% CO2 for culture, adding 50ml of immune cell culture medium in a T82 bottle on the third day, putting cells in a T82 cell bottle into a cell bag for culture on the sixth day, adding 100ml of cell culture solution in the A bottle at the same time, adding 150ml of immune cell culture solution in the immune cell bag on the ninth day, and adding B immune cell culture solution in the immune cell bag on the twelfth day. Supplementing cell culture solution after three days, wherein the number of immune cells can reach 1.6x 1010 cells after the immune cells are cultured for 15 days;

sixth: after the cell bag is cultured, placing the cell bag into a 500ml conical centrifuge bottle, balancing and centrifuging at 2600r/min for 15min, after the centrifugation is finished, removing the supernatant, adding 40ml of 0.9% sodium chloride into the centrifuge bottle after the supernatant is removed, blowing the sodium chloride by using a bus dropper, completely blowing the cell sediment at the bottom of the bottle into the sodium chloride solution, balancing and placing the cell bag into a centrifuge at 2600r/min for 15min, removing the supernatant from two 500ml centrifuge bottles after the centrifugation, and adding 20ml of 0.9% sodium chloride solution into each centrifuge bottle for suspending the cells for later use.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention relates to a high-efficiency human blood immune cell CD4T separation culture technology, which has the advantages of improving the working efficiency, saving the working time, high immune cell group yield, convenient use and low material consumption, and the whole process of self-blood sample injection into a blood collection tube, serum separation and blood sample preservation is carried out in the same branch tube, thereby avoiding the blood sample from polluting operators, preventing the infection of virus in the blood, reducing the medical waste and improving the working efficiency.

Drawings

FIG. 1 is a table showing the detection results of a five-classification blood cell detector according to the present invention;

FIG. 2 is a table comparing the extraction of mononuclear cells and T lymphocyte separation fluid from the separation gel of the present invention;

FIG. 3 is a schematic diagram showing the proportions of CD4, CD56+ CIK, NK, NKT and cells before and after cell expansion by flow cytometry.

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. 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.

Referring to fig. 1-3, the present invention provides a technical solution of a high efficiency separation and culture technique for human blood immune cells CD 4T:

a high-efficiency separation culture technology of human blood immune cells CD4T is characterized in that the separation culture of CD4T comprises a separation method of mononuclear cell immune cells in human blood and an in vitro culture amplification method of mononuclear cells in human blood, and the separation culture technology comprises the following steps:

firstly, the method comprises the following steps: the instrument for preparing the separated blood comprises three parts, namely a centrifuge tube, an anticoagulant, a separation gel, and an immunocyte separation gel, wherein the immunocyte separation gel is a hydrophobic organic compound, and the action principle is as follows: the serum specific gravity is about 1.02, and the red blood cell specific gravity is: 093, polymorphonuclear leukocyte specific gravity: 1.092, platelet specific gravity is about: 1.032, the specific gravity of the mononuclear cells is as follows: 1.076-1.090, maintaining the specific gravity of the separation gel at 1.77, forming an isolation layer among serum, white blood cells and red blood cells by the immune cell separation gel under the action of centrifugal force, placing 5ml of the separation gel at the bottom of a 50ml centrifuge tube, sequentially adding polyolefin, polyester and propylene into the centrifuge tube, centrifuging for 3500r/min, centrifuging for 15min, and preparing the centrifuge tube with the immune cell separation gel for later use;

secondly, the method comprises the following steps: extracting 2.0ml heparin anticoagulant by using a 50ml syringe, pushing and pulling the syringe several times to enable heparin to wet the wall of the syringe, replacing a new syringe needle, extracting 40ml peripheral venous autoblood, placing 40ml anticoagulant whole blood into a centrifuge tube made of 50ml separation gel, leveling the centrifuge tube, 3500r/min, centrifuging the blood for 15 minutes to obtain separation gel, namely forming an isolation layer among serum, white blood cells and red blood cells, keeping the serum in the original state, keeping the serum unchanged, enabling the isolation layer to be tightly adhered to the wall of the centrifuge tube, avoiding the influence of fibrin and hemolysis, adhering a white membrane layer on the upper surface of the separation gel, and enabling the white membrane layer to be a target cell layer;

To summarize the experiment: the result is not ideal by a method of separating and extracting immune cell groups by using T lymphocyte extracting solution twice and autologous 9ml whole blood, and the cells are not detected and counted by using a five-classification blood cell detector twice, which indicates that the number of the cells is too small and the system cannot count. By another test blood cell tester, a minute amount of total mononuclear immune cell population is detected. The method for separating the mononuclear cells by using the T lymphocyte separation liquid is effective, cannot uniformly separate and extract quality standards, has complicated operation steps, generally has the time for separating one part of blood by operation for about 2 and a half hours, has large influence on the quality of target extraction by the technology of operators and the amount of the extracted blood, and has low target cell yield. Five classifications the other method detects 3 times of concentrated single cells twice, detects trace amount, and respectively comprises the following steps: (MN1.243, 19% of Whole blood mononuclear cells extracted%) (MN3.378. 39% of Whole blood mononuclear cells extracted.)

The MN is the total number of the mononuclear cells, about 2 times of the mononuclear immune cell amount is lost as a result, the immune cell group (average of two times) obtained by the existing separation technology method only accounts for 30% of the whole blood immune cells, and 70% of the immune cell group is lost.

Examples

Experimental materials:

equipment: 15ml conical centrifuge tube, separation rubber tube, suction tube, sterile drying syringe, sterile cotton ball, rubber tourniquet, horizontal centrifuge, biological microscope, five-classification blood cell analyzer, etc.

Immune cell separation gel: the immune cell separating gel is a viscous fluid, and the structure of the immune cell separating gel contains a large number of hydrogen bonds, and a network structure is formed due to the association of the hydrogen bonds. Under the action of centrifugal force, the net structure is broken down and becomes a fluid with low viscosity. When the centrifugal force disappears, the network structure is formed again, and the high-viscosity fluid is recovered, and the property is called thixotropy, and the immune cell separation gel can be prepared by utilizing the property. When the immune cell separation gel and anticoagulated blood are centrifuged in the same test tube, the immune cell separation gel forms a colloidal separation layer between serum and red blood cells to separate the serum, the white blood cells and the red blood cells. Thereby improving the yield of immune cells and improving the working efficiency.

The immune cell separation gel is a hydrophobic organic compound, and the action principle is as follows: generally, the specific gravity of serum is about 1.02, the specific gravity of blood clot is about 1.08, and the specific gravity of separation gel is maintained at 1.05. Under the action of centrifugal force, the immune cell separating gel forms an isolating layer between serum and white blood cells and between red blood cells. Immune cell separating gel, polyolefin, polyester and propylene, wherein the separating gel is placed at the bottom of a test tube, a blood sample is injected into the test tube, the test tube is placed in a centrifugal machine for centrifugation, and the separating gel forms an isolating layer between serum and a blood clot. And the serum is kept unchanged. The barrier layer adheres tightly to the wall of the tube and also avoids fibrin and hemolysis effects.

Reagent:

heparin solution, prepared with physiological saline to 500u/ml

Salt water

2g/L trypan blue staining solution.

Sample heparin anticoagulated human peripheral venous blood.

The experimental method comprises the following steps:

0.5ml heparin anticoagulant is extracted by a 10ml syringe, the syringe is pushed and pulled for several times to wet the wall of the syringe, a new syringe needle is replaced, and 11ml peripheral venous autoblood is extracted.

11ml of anticoagulated whole blood is injected into a 2ml negative pressure blood collection tube for testing, and the remaining 9ml of anticoagulated whole blood is placed in a centrifuge tube with 15ml of separation gel.

The mixture is balanced and centrifuged for 15 minutes at 3500 r/min.

Taking out the centrifuge tube, discarding the supernatant to the middle layer, reserving 3ml of plasma, gently and repeatedly blowing and beating the plasma to the surface of the separation gel by using a suction tube, completely blowing the white membrane into the plasma, putting the blown and beaten plasma into a new centrifuge tube, and preparing for detection.

Or cleaning for 1-2 times, transferring the separated cells into a 50ml centrifuge tube, adding 30ml of saline, blowing with a suction tube, balancing with a centrifuge, and centrifuging at 2200r/min for 20 min.

Taking out the centrifuge tube, removing the supernatant, and continuing the subsequent operation.

The experimental results are as follows:

a triple concentrated solution was prepared by separating and extracting mononuclear cells with a separation gel, and was compared with raw blood by a five-classification resistance method, as shown in FIG. 1.

The obtained mononuclear cells were isolated for quality testing: the mononuclear cells separated by the separation gel are subjected to bacterial culture, activity detection, endotoxin residue, eluate detection and the like, as shown in FIG. 2.

In vitro culture expansion of mononuclear cells from 45ml of certified whole blood: taking out the centrifuge tube, discarding the supernatant to the middle layer, reserving 15ml of plasma, gently and repeatedly blowing and beating the plasma to the surface of the immune cell separation gel by using a suction tube, completely blowing the white membrane on the separation gel into the plasma, putting the blown and beaten plasma into a new centrifuge tube, cleaning for 1-2 times, transferring the separated cells into a 50ml centrifuge tube, adding 30ml of saline, blowing and beating by using the suction tube, balancing by using a centrifuge, and centrifuging in the centrifuge for 2200r/min and rotating for 20 min.

The immune cell culture solution is prepared by adding 8% fetal calf serum into 1000ml of immune cell culture medium of KANGNING brand in bottle A, adding 200 million U of interleukin 2 in bottle A, adding 10mg of thymopentin, adding 500 million U of interferon a2B1m, culturing for 12 days, and adding immune cell culture solution in bottle B: 1000ml of corning brand immune cell culture medium is added with 8 percent fetal calf serum, 200 million U of interleukin 2 in two bottles, 10mg of thymopentin, 500 million U of interferon a2b1m and 0.25g of BCG. To stimulate induction of monocytes.

Taking out the centrifuge tube, removing supernatant, leaving precipitate, adding 20ml of immune cell culture solution in an A bottle into the precipitated tissue, repeatedly blowing and beating the precipitate by using a bus dropper to completely blow off the precipitate, transplanting the precipitate into a T82 cell culture bottle after blowing, putting a T82 cell culture bottle into an incubator of 5% CO2 for culture, adding 50ml of immune cell culture medium in a T82 bottle on the third day, putting cells in a T82 cell bottle into a cell bag for culture on the sixth day, adding 100ml of cell culture solution in the A bottle at the same time, adding 150ml of immune cell culture solution in the immune cell bag on the ninth day, and adding B immune cell culture solution in the immune cell bag on the twelfth day.

The cell growth state was observed every day, and cell counting was performed every 4 days. And supplementing cell culture solution after supplementing interleukin 2(50 ten thousand u) for three days according to the growth state of the cells. The number of immune cells can reach 3.5X 109 cells after 14 days of immune cell culture. The culture was terminated at 8.5X 109 on day 21.

Table of cell number, amplification multiple and survival rate before and after amplification

After the cell bag culture is finished, the cell bag is placed into a 500ml conical centrifuge bottle, and the cell bag is subjected to balance centrifugation 2600r/min and rotated for 15 min. And after centrifugation, discarding the supernatant, adding 40ml of 0.9% sodium chloride into a centrifuge bottle, blowing and beating the mixture by using a bus dropper, completely blowing and scattering cell sediment at the bottom of the bottle into a sodium chloride solution, balancing, putting the bottle into a centrifuge 2600r/min, rotating for 15min, removing the supernatant by using two 500ml centrifuge bottles after centrifugation, and adding 20ml of 0.9% sodium chloride solution into each centrifuge bottle for suspension, wherein the suspension is reserved.

The flow cytometry detection method comprises the following steps: cell samples obtained after 21 days of culture in peripheral blood mononuclear cells isolated on the same day as in example 1 were taken at 6X106 cells/tube and washed 1 time with PBS. Adding flow detection antibodies respectively for double-labeled flow phenotype detection: FITC-labeled murine anti-human CD4 antibody and APC-labeled murine anti-human CD56 antibody; after waiting for 20 minutes at room temperature, the cells were washed 1 time with PBS and analyzed by flow cytometry.

The results are shown in FIG. 3, after 21 days of induction culture, CD4+ CD56+ and CIK/NK/NKT group immune cells increased from about 11.7% on day 0 (FIG. 3CD 4A), and after 21 days of culture, CD4+ CD56+ and CIK/NK/NKT group immune cells increased to 87% (FIG. 3CD 4B).

Example cell killing Activity assay

Taking K562 human chronic myelogenous leukemia cells in logarithmic growth phase as target cells, proportioning the cell density to be 7x105 cells/ml, and laying 0.1ml in each hole of a 48-hole culture plate. The CD4 immune cell populations on day 14 and day 21 obtained in example 6 of the present invention were taken, adjusted to densities of 2X 106/ml, 8X 106/ml, 1.6X 107/ml, and 3.2X 107/ml, and added to 48-well culture plates at 0.1ml per well so that the effective-to-target ratios were 1:1, 5:1, 10:1, and 20:1, and each set was provided with three duplicate wells in the following manner:

blank group (a): AIM-V-DMEM (0.2ml)

(B) The method comprises the following steps CD4 immune cell population (0.1ml) + AIM-V-DMEM (0.1ml)

Control group (C): k562(0.1ml) + AIM-V-DEEM (0.1ml)

Experimental group (D): k562(0.1ml) + CD4 immune cell population (0.1ml)

After the inoculated cells are CO-cultured for 48 hours at 37 ℃ under the condition of 5% CO2, 20 microliters of CCK-8 reagent is added into each hole, the mixture is shaken up, the cells are continuously cultured for 5 hours at 37 ℃ under the condition of 5% CO2, and the absorbance is measured at the wavelength of 450nm by an enzyme-labeling instrument.

The killing activity was calculated as follows: the result shows that the CD4 immune cell population prepared by the invention has high killing activity on a tumor K562 cell strain, and the killing rate can reach 100% at the 14 th day under the effect-target ratio of 10:1 and 20: 1; the killing rate can reach more than 90% at the 14 th day and 100% at the 21 st day under the effective target ratio of 5: 1; the kill rate was less than 40% at both day 14 and day 21 at an effective target ratio of 1:1, but the kill rate was significantly higher at day 21 than at day 14.

Killing activity of CD4 immune cell population against tumor K562 cells table:

effective target ratio	1：1	5：1	10：1	20：1
					Day 14	26％	90％	100％	100％
Day 21	40％	100％	100％	100％

In the description of the present invention, it is to be understood that the indicated orientations or positional relationships are based on the orientations or positional relationships shown in the drawings and are only for convenience in describing the present invention and simplifying the description, but are not intended to indicate or imply that the indicated devices or elements must have a particular orientation, be constructed and operated in a particular orientation, and are not to be construed as limiting the present invention.

In the present invention, unless otherwise explicitly specified or limited, for example, it may be fixedly attached, detachably attached, or integrated; can be mechanically or electrically connected; the terms may be directly connected or indirectly connected through an intermediate, and may be communication between two elements or interaction relationship between two elements, unless otherwise specifically limited, and the specific meaning of the terms in the present invention will be understood by those skilled in the art according to specific situations.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims

1. A high-efficiency separation culture technology of human blood immune cells CD4T is characterized in that the separation culture of CD4T comprises a separation method of mononuclear cell immune cells in human blood and an in vitro culture amplification method of mononuclear cells in human blood, and the separation culture technology comprises the following steps:

thirdly, the steps of: taking out the centrifuge tube, discarding the supernatant to the middle layer, reserving 15ml of plasma, gently and repeatedly blowing and beating the plasma to the surface of the immune cell separation gel by using a suction tube, completely blowing a white membrane on the separation gel into the plasma, putting the blown and beaten plasma into a new centrifuge tube, cleaning for 1-2 times, transferring the separated cells into a 50ml centrifuge tube, adding 30ml of saline into the centrifuge tube, blowing and beating by using the suction tube, balancing by using a centrifuge, and centrifuging in the centrifuge for 2200r/min and rotating for 20 min;

fourthly: the immune cell culture solution is prepared by adding 8% fetal calf serum into 1000ml of immune cell culture medium in bottle A, adding 200 ten thousand U of interleukin 2, adding 10mg of thymopentin, adding 500 ten thousand U of interferon a2B1m, and adding immune cell culture solution in bottle B after 12 days of culture: adding 8% fetal calf serum into 1000ml immune cell culture medium, adding 200 ten thousand U of interleukin 2 two bottles, adding 10mg of thymopentin, adding 500 ten thousand U of interferon a2b1m, and adding 0.25g of BCG;