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CN112251407A - Amplification culture method of umbilical cord blood CIK cells - Google Patents

️Fri Jan 22 2021

CN112251407A - Amplification culture method of umbilical cord blood CIK cells - Google Patents

Amplification culture method of umbilical cord blood CIK cells Download PDF

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Publication number

CN112251407A

CN112251407A CN202011209454.1A CN202011209454A CN112251407A CN 112251407 A CN112251407 A CN 112251407A CN 202011209454 A CN202011209454 A CN 202011209454A CN 112251407 A CN112251407 A CN 112251407A Authority

China

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culture

cells

cord blood

cell

amplification

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2020-11-03

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Pending

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CN202011209454.1A

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Chinese (zh)

Inventor

陈海佳

王小燕

简毅超

张梦晨

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Guangzhou Sailaila Biological Gene Engineering Co ltd

Guangzhou Kangqilai Precision Medical Technology Co ltd

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Guangzhou Sailaila Biological Gene Engineering Co ltd

Guangzhou Kangqilai Precision Medical Technology Co ltd

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2020-11-03

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2020-11-03

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2021-01-22

2020-11-03 Application filed by Guangzhou Sailaila Biological Gene Engineering Co ltd, Guangzhou Kangqilai Precision Medical Technology Co ltd filed Critical Guangzhou Sailaila Biological Gene Engineering Co ltd

2020-11-03 Priority to CN202011209454.1A priority Critical patent/CN112251407A/en

2021-01-22 Publication of CN112251407A publication Critical patent/CN112251407A/en

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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
- C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2301—Interleukin-1 (IL-1)
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2302—Interleukin-2 (IL-2)
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/24—Interferons [IFN]
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/50—Cell markers; Cell surface determinants
- C12N2501/515—CD3, T-cell receptor complex

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Abstract

The invention relates to the technical field of cell culture, in particular to an amplification culture method of an umbilical cord blood CIK cell. The invention discloses an expansion culture method of cord blood CIK cells, which is characterized in that a cell culture bag is applied to CIK cell induction culture of cord blood separation for the first time, and the cell culture bag has the advantages of large culture medium volume and large gas exchange area, so that red blood cells mixed with cord blood mononuclear cells can be diluted in the culture process, the rapid metabolic removal of the red blood cells is promoted, the influence of the red blood cell mixing on the culture effect is avoided, and the purity of the CIK cells is improved. The cell culture bag is simple and convenient to operate, and the aim of quickly activating and amplifying the CIK cells in a short time is also fulfilled.

Description

Amplification culture method of umbilical cord blood CIK cells

Technical Field

The invention relates to the technical field of cell culture, in particular to an amplification culture method of an umbilical cord blood CIK cell.

Background

The CIK cells are a group of heterogeneous cells in a human body, have the capacity of efficiently killing infected and diseased cells, are not activated due to physiological reasons only in weak and sub-healthy people, and are quickly activated to attack and eliminate abnormal cells after being amplified in vitro and returned to the human body.

At present, the cord blood is separated from the cells mainly according to the difference of cell density by means of the gravity acceleration generated by centrifugation and the separation and purification of the cells. The cord blood has a large number of nucleated cells compared with adult peripheral blood, is rich in hematopoietic stem cells, and has large volume and low density of red blood cells in the cord blood. The density of the common lymphocyte separation liquid is between 1.075 and 1.090, the method is mainly suitable for peripheral blood separation, and when the method is applied to separation of umbilical cord blood mononuclear cells, the problems of red blood cell mixing and unobvious layering occur, and the effect of CIK cell culture is interfered.

Disclosure of Invention

The invention provides an amplification culture method of cord blood CIK cells, which solves the problems that red blood cells are mixed and CIK cell culture is interfered when a conventional density gradient centrifugation method is adopted to separate cord blood mononuclear cells.

The specific technical scheme is as follows:

the invention provides an expansion culture method of an umbilical cord blood CIK cell, which comprises the following steps:

step 1: on

day

0, inoculating umbilical cord blood mononuclear cells into a cell culture bag containing IFN-gamma and a basic culture medium for culture;

step 2: after culturing for 24 h-26 h, adding OKT-3, IL-1 alpha and IL-2 for continuous culture;

and step 3: observing the cell culture state every day, counting the cells every 2-3 days, if the cells are proliferated, supplementing IL-2 and continuously culturing the cells by using a basic culture medium, and if the cells are not proliferated, supplementing IL-2 on the 4 th day;

and 4, step 4: and 3, after the first fluid infusion, supplementing IL-2 and a basic culture medium every 2-3 days for continuous culture till 14-21 days, and harvesting the cord blood CIK cells.

IFN-gamma: type II interferons, also known as IFN-gamma or immunointerferons, are produced by mitogens stimulating T lymphocytes. Interferon is a highly effective antiviral bioactive substance, and is also a lymphokine with a wide range of immunoregulatory actions.

OKT-3: the monoclonal antibody against CD3 (OKT3) against CD3 is capable of stimulating or blocking T cell activation signaling, eliminating effector T cells or inducing the production of regulatory T cells. Has effects in activating T cell proliferation and activation.

IL-1. alpha.: interleukin-1, can activate CD4+ T cells, promote B cell growth and differentiation and IL-2 or interferon cooperate and can enhance NK cell activity.

IL-2: also known as interleukin-2, is a cytokine in the chemokine family. Has the cell factor of activating T cell to proliferate and promoting the growth, proliferation and differentiation of lymphocyte.

In the invention, the cell culture bag has the advantages of large culture medium volume and large gas exchange area, and can dilute the red blood cells mixed with the cord blood mononuclear cells in the culture process, promote the rapid metabolic removal of the red blood cells, and avoid the influence of the red blood cells mixed on the culture effect, thereby improving the purity of the CIK cells. The invention adopts the cell culture bag for culture, omits the step of protein coating of a culture container, has simple and convenient operation of the cell culture bag, and also achieves the aim of quickly activating and amplifying the CIK cells in a short time (14 d-21 d).

In step 1 of the invention, the separation of cord blood mononuclear cells comprises the following steps:

adding the diluted cord blood into lymphocyte separation liquid, centrifuging for 30min at 800g, sucking a leucocyte layer for resuspension, and centrifuging for 10min at 600g to obtain cord blood mononuclear cells;

in the invention, the cord blood is freshly collected for no more than 24 hours, the cord blood is weighed and transferred to a centrifuge tube, a buffer solution with the same volume as the cord blood is sucked and added into the centrifuge tube to be uniformly mixed, so as to obtain diluted cord blood, and the buffer solution is preferably PBS, normal saline or D-PBS buffer solution; more preferably, the ratio of 2: 1, adding the mixture into a centrifugal tube containing a lymphocyte separation solution to form a clear interface between the two, and centrifuging the mixture for 30min at 800g preferably; after centrifugation, the solution in the centrifugal tube is divided into 4 layers, namely a plasma layer, a tunica albuginea layer (CBMC layer), a lymphocyte separation liquid layer, red blood cells and a granulocyte layer in sequence, the suspension containing CBMC is sucked out and added into the centrifugal tube, preferably, the buffer solution is added for resuspension, the centrifugal tube is centrifuged for 10min at 600g, and the supernatant is discarded to obtain the umbilical cord blood mononuclear cells.

The density of a common lymphocyte separation liquid is between 1.075 and 1.090, so that the lymphocyte separation liquid is mainly suitable for peripheral blood separation, and the conventional centrifugation parameter is 2000rpm and the centrifugation is 20 min. The cord blood has a large number of nucleated cells compared with adult peripheral blood, is rich in hematopoietic stem cells, and has large volume and low density of red blood cells in the cord blood. When the conventional density gradient centrifugation method is applied to umbilical cord blood separation, the problems of red blood cell mixing and unobvious layering occur, and the effect of next cell culture is influenced by interference. The method adopts specific centrifugation parameters of 800g for centrifugation for 30min when separating cord blood mononuclear cells, is favorable for layering of cells and reduces the condition of red blood cell mixing, and further adopts specific centrifugation parameters of 600g for centrifugation for 10min in the cell washing process, thereby being further favorable for layering of cells.

It should be noted that the conventional umbilical cord blood mononuclear cells are seeded at a density of (1.0-2.0). times.10⁶The inoculation density of the cord blood mononuclear cells is improved to 1.5-3.0 multiplied by 10 after the cell culture bag is used in the invention⁶one/mL, preferably 2.0

10⁶and/mL, thereby facilitating proliferation of CIK cells.

In step 1 of the present invention, the final concentration of IFN- γ in the culture system is 500-1500U/mL, preferably, more preferably 1000U/mL.

In the step 2 of the invention, after culturing for 24-26 h (marked as day 1), OKT-3, IL-1 alpha, IL-2 and a basal medium are added for continuous culture; the OKT-3, the IL-1 alpha and the IL-2 are added simultaneously within 24h, 24.5h, 25h, 25.5h or 26h of culture;

the final concentrations of the OKT-3, the IL-1 alpha and the IL-2 in the culture system are respectively 30-70ng/mL, 80-120U/mL and 150-500U/mL, preferably 40-60 ng/mL, 90-110U/mL and 150-300U/mL, more preferably 50ng/mL, 100U/mL and 200U/mL.

The step 3 of the invention is specifically as follows: culturing for 3-4 days, preferably culturing for 3 days, if the cells obviously proliferate, supplementing IL-2 and a basal medium, if the cells do not obviously proliferate, supplementing IL-2 on the 4 th day of culturing, and ensuring the cell density to be (2.0-4.5) multiplied by 10 in the whole culturing process⁶Per mL;

if the cells obviously proliferate, the final concentration of the IL-2 supplement in the culture system is 200-600U/mL, preferably 200U/mL; if the cells do not proliferate significantly, the final concentration of the supplemented IL-2 in the culture system is 300-800U/mL, preferably 200U/mL.

In step 4 of the invention, after the first fluid replacement is performed in step 3, IL-2 is supplemented every 2-3 days, preferably every 2 days, and the culture is continued with the basal medium, the final concentration of IL-2 in the culture system is 800U/mL, preferably 400U/mL, and more preferably 350U/mL when the umbilical cord blood CIK cells are harvested after the culture is cultured for 14-21 days, preferably 18 days.

In the initial stage of cell culture, namely in the step 1 and the step 2, the addition amount of the cell factors is less, and the amounts of the cell factors are gradually increased in the step 3 and the step 4, so that the aim of rapidly proliferating the CIK cells is fulfilled.

In the present invention, the final concentration of the fetal calf serum in the culture systems of step 1, step 2, step 3 and step 4 is 2-8 vol%, preferably 4.5 vol%.

In the present invention, the culture is preferably performed in 5% CO₂Culturing in a carbon dioxide incubator with the temperature of 37 ℃ and the saturation humidity of 95 percent;

the basic culture medium is a serum-free culture medium containing fetal calf serum;

the serum-free culture medium is selected from GT-T551 culture medium, DWK culture medium, Lonza culture medium or X-VIVO15 culture medium.

In the CIK cell culture process, the cell culture system can be cultured in bags according to the volume of the cell culture system and the culture proliferation condition, and the volume of the cell culture system can be 1200ml, 1250ml, 1300ml, 1350ml, 1400ml, 1450ml, 1500ml, 1550ml, 1600ml, 1650ml, 1700ml, 1750ml or 1800 ml.

According to the technical scheme, the invention has the following advantages:

the invention provides an expansion culture method of CIK cells in umbilical cord blood, which is characterized in that a cell culture bag is applied to CIK cell induction culture of umbilical cord blood separation for the first time, and the cell culture bag has the advantages of large culture medium volume and large gas exchange area, so that red blood cells mixed with mononuclear cells in the umbilical cord blood can be diluted in the culture process, the rapid metabolism removal of the red blood cells is promoted, the influence of the red blood cells mixed on the culture effect is avoided, and the purity of the CIK cells is improved. The cell culture bag is simple and convenient to operate, and the aim of quickly activating and amplifying the CIK cells in a short time (14 d-21 d) is fulfilled.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without inventive exercise.

FIG. 1 is a diagram of CIK cell proliferation of umbilical cord blood provided in example 2 of the present invention;

figure 2 is a flow cytometric image provided in example 2 of the present invention.

Detailed Description

In order to make the objects, features and advantages of the present invention more obvious and understandable, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it should be apparent that the embodiments described below are only a part of the embodiments of the present invention, and not all 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.

Example 1 cord blood mononuclear cell CBMC isolation

(1) 85mL of freshly collected (no more than 24h) cord blood was weighed and transferred to a centrifuge tube. And (3) sucking normal saline with the same volume as the blood, adding the normal saline into a centrifugal tube, diluting the blood and uniformly mixing. Taking a plurality of centrifuge tubes, adding the lymphocyte separation solution into each tube by using a pipette, and then adding the diluted blood into each tube by using the pipette according to the ratio of 2: 1 was slowly added to the surface of the lymphocyte separation medium to form a clear interface between the two. The tube was trimmed and carefully placed in a centrifuge and centrifuged at 800g for 30 min.

(2) After centrifugation, the centrifuge tube was carefully taken out, and was divided into 4 layers from top to bottom, namely a plasma layer, a tunica albuginea layer (CBMC layer), a lymphocyte separation liquid layer, and a red blood cell and granulocyte layer. The upper CBMC-containing suspension was aspirated and transferred to a new 50mL centrifuge tube.

(3) The appropriate amount of RPMI-1640 medium was added to the centrifuge tube by pipette to resuspend CBMC and centrifuge at 600g for 10 min. After centrifugation, the supernatant was discarded and repeated once. Adding a proper amount of serum-free culture medium, and fully mixing the cells to obtain the CBMC cell number of 3.79 multiplied by 108.

Example 2 CIK cell induced expansion

(1) The CBMC isolated in example 1 was added with 2% fetal bovine serum in X-VIVO15 serum-free medium to resuspend the cell pellet at

10⁶The cells are inoculated in a cell culture bag at the inoculation density of one cell/mL, and 1000U/mL IFN-gamma is added according to the amount and mixed evenly. Shift to 5% CO₂And culturing in a carbon dioxide incubator with the saturation humidity of 95% at 37 ℃.

(2) After 24h of culture (day 1), it was supplemented with 50ng/mL OKT-3, 100U/mL IL-1. alpha. and 200U/mL IL-2.

(3) The color and cell status of the culture broth were observed daily, and samples were taken every 2 days for cell counting. According to the cell proliferation condition, the culture medium of X-VIVO15 containing 2% fetal calf serum is supplemented properly, and meanwhile, 200U/mL IL-2 is added according to the total amount, so that the cell density in the whole culture process is ensured to be 2.0-4.5

10⁶In the size per mL range.

(4) Cells were collected by centrifugation during 18d cell culture, and cell count, cell viability assay and flow cytometry assay were performed, with the results shown in fig. 1 and 2.

FIG. 1 is a diagram of the proliferation of CIK cells in umbilical cord blood provided in this example. The results in fig. 1 show that the cells proliferated approximately 27-fold within 18 days.

Fig. 2 is a flow cytometric image provided for this example. The results in fig. 2 show that the total T lymphocytes (CD3+) were 99.96% and the NKT cells (CD3+ CD56+) ratio was 34.19%.

Example 3

95mL of umbilical cord blood from another volunteer was collected, and the number of CBMC cells was counted by the same CMBC isolation method as in example 1 to obtain 2.00X 10^8, and the CIK culture method as in example 1 was used. Cells were harvested at 18d, showing that the cells proliferated about 55-fold within 18 days; the total T lymphocytes (CD3+) accounted for 99.30% and the NKT cells (CD3+ CD56+) ratio was 20.37%.

The above-mentioned embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the same; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

Claims (10)

1. An amplification culture method of cord blood CIK cells is characterized by comprising the following steps:

step 1: on day 0, inoculating umbilical cord blood mononuclear cells into a cell culture bag containing IFN-gamma and a basic culture medium for culture;

step 2: after culturing for 24 h-26 h, adding OKT-3, IL-1 alpha and IL-2 for continuous culture;

and 4, step 4: and 3, after the first fluid infusion, supplementing IL-2 and a basic culture medium every 2-3 days for continuous culture till 14-21 days, and harvesting the cord blood CIK cells.

2. The amplification culture method according to claim 1, wherein the amount of the umbilical cord blood mononuclear cells to be inoculated is 1.5 to 3.0X 10⁶one/mL.

3. The amplification culture method of claim 1, wherein the final concentration of IFN- γ in the culture system in step 1 is 500-;

step 2, the final concentrations of the OKT-3, the IL-1 alpha and the IL-2 in the culture system are respectively 30-70ng/mL, 80-120U/mL and 150-500U/mL;

step 4 the final concentration of the IL-2 in the culture system is 250-800U/mL.

4. The amplification culture method according to claim 1, wherein the basal medium is a serum-free medium containing fetal bovine serum;

the serum-free culture medium is selected from GT-T551 culture medium, DWK culture medium, Lonza culture medium or X-VIVO15 culture medium.

5. The amplification culture method of claim 1, wherein in step 2, OKT-3, IL-1 α and IL-2 are added simultaneously within 24h, 24.5h, 25h, 25.5h or 26h of culture.

6. The amplification culture method of claim 4, wherein the final concentration of the fetal calf serum in the culture systems of step 1, step 2, step 3 and step 4 is 2-8 vol%.

7. The amplification culture method according to claim 1, wherein the step 3 specifically comprises: culturing for 3-4 days, if the cells proliferate, supplementing IL-2 and a basal medium, and if the cells do not proliferate, supplementing IL-2 on the 4 th day of culturing.

8. The amplification culture method according to claim 7, wherein the final concentration of IL-2 in the culture system is 200-600U/mL if the cells are proliferated;

if the cells are not proliferated, the final concentration of the supplemented IL-2 in the culture system is 300-800U/mL.

9. The amplification culture method according to claim 1, further comprising, before step 1: separating umbilical cord blood mononuclear cells;

the separation specifically comprises the following steps:

and adding the diluted cord blood into lymphocyte separation liquid, centrifuging for 30min at 800g, sucking a leucocyte layer for resuspension, and centrifuging for 10min at 600g to obtain the cord blood mononuclear cells.

10. The amplification culture method of claim 1, wherein the cell density is maintained at 2.0-4.5X 10 during the culture⁶one/mL.

CN202011209454.1A 2020-11-03 2020-11-03 Amplification culture method of umbilical cord blood CIK cells Pending CN112251407A (en)

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