A method for culturing highly active γδT cells in vitro

Abstract

The present invention proposes a method for culturing highly active γδT cells in vitro, comprising adding a sea cucumber polysaccharide extract to the culture medium on the second to third day of in vitro culture, replacing the culture medium on the fourth to fifth day of in vitro culture, and simultaneously adding the sea cucumber polysaccharide extract. The γδT cells obtained by the present invention have high cytotoxic activity against Daudi lymphoma cells, and the cytotoxic activity increases to varying degrees with prolonged culture time, resulting in highly active γδT cells. The present invention has high efficiency in amplifying γδT cells, facilitates the rapid acquisition of highly active γδT cells, and is simple and convenient to operate.

Description

Technical Field

[0001] The present invention relates to the field of cell culture, and in particular to an in vitro culture method for highly active γδT cells. Background Art

[0002] Based on the differences in their T cell surface receptors (TCRs), T cells can be divided into αβT cells and γδT cells. αβT cells account for over 95% of all T cells, while γδT cells only account for 2-5% of the total T cell population and are primarily distributed in mucosa-associated lymphoid tissues. Previous research has limited understanding of the functions and mechanisms of γδT cells. However, with the rapid advancement of biotechnology, the physiological functions and mechanisms of action of γδT cells have been continuously uncovered. Studies have found that the non-MHC-restricted tumor cell killing properties of γδT cells make them an ideal cell preparation material. They can directly initiate tumor cell killing without the presence of antigen-presenting cells (APCs) and are also highly effective against tumor cells that express low MHC molecules and have a tendency to escape the immune system.

[0003] γδT cells display cytotoxic activity against a variety of tumor cell types, and certain clinical approaches initially developed to enhance T cell cytotoxicity may also activate γδT cells. Furthermore, a growing number of studies have identified novel approaches to enhance γδT cell anti-tumor immunity and expand ex vivo NK cell populations, paving the way for a new generation of anti-cancer immunotherapies. Over the past decade, over 60,000 γδT cell treatments have been administered, and aside from occasional adverse reactions such as fever, no side effects requiring treatment have occurred, demonstrating the clinical safety of γδT cell therapy. γδT cell therapy is often combined with dendritic cell therapy, hyperthermia, and low-dose chemotherapy, and has been observed to prolong survival in patients with advanced pancreatic cancer and enhance the therapeutic efficacy of breast cancer.

[0004] Currently, one of the current research hotspots is to obtain high-purity and high-activity γδT cells with higher efficiency, as well as the research on the drugability of γδT cells, while there are fewer studies on the effects of γδT cells on lymphoma. Summary of the Invention

[0005] In view of this, the present invention proposes an in vitro culture method for highly active γδT cells. The obtained γδT cells have high killing activity against lymphoma Daudi cells. With the extension of culture time, the killing activity increases to varying degrees, providing a certain data basis for the killing activity of γδT cells cultured in vitro against lymphoma Daudi cells.

[0006] The technical solution of the present invention is achieved as follows:

[0007] The invention discloses an in vitro culture method for highly active γδT cells, comprising adding sea cucumber polysaccharide extract to a culture medium on the 2nd to 3rd day of in vitro culture, replacing the culture medium on the 4th to 5th day of in vitro culture, and adding the sea cucumber polysaccharide extract at the same time.

[0008] It is further described that the preparation method of the sea cucumber polysaccharide extract includes adding ethanol to sea cucumber powder, ultrasonic extraction, standing, discarding the supernatant, taking precipitate 1, adding Sevag reagent for extraction, collecting the supernatant, alcohol precipitation, discarding the supernatant, centrifuging, taking precipitate 2, freeze-drying, dissolving in water, passing through an HP-SAX strong anion exchange chromatography column, collecting the eluate, concentrating, dialyzing, and freeze-drying to obtain the sea cucumber polysaccharide extract.

[0009] It is further explained that the mass volume ratio of sea cucumber powder and ethanol is 1:20-25 mL; Sevag reagent is chloroform and n-butanol in a volume ratio of 4:1; and the HP-SAX strong anion exchange chromatography column is eluted with 0.4 M, 0.6 M, and 0.8 M NaCl solutions, respectively.

[0010] It is further explained that on the 2nd to 3rd day of in vitro culture, the O2 volume concentration is adjusted to 75-80%, and the rest is CO2. On the 4th to 5th day of in vitro culture, the O2 volume concentration is adjusted to 85-90%, and the rest is CO2.

[0011] Further explanation: when changing the culture medium, adjust the cell density to 4-5×10 5 pieces / mL.

[0012] It is further explained that the added amount of sea cucumber polysaccharide extract is 20 to 40 μg / mL.

[0013] It is further explained that the amount of sea cucumber polysaccharide extract added on the 2nd to 3rd day of in vitro culture is 30 to 40 μg / mL, and the amount of sea cucumber polysaccharide extract added on the 4th to 5th day of in vitro culture is 20 to 30 μg / mL.

[0014] It is further explained that the amount of sea cucumber polysaccharide extract added during the 2nd to 3rd day of in vitro culture is 38 μg / mL, and during the 4th to 5th day of in vitro culture, the amount of sea cucumber polysaccharide extract added is 22 μg / mL.

[0015] The present invention provides a sea cucumber polysaccharide extract capable of improving the activity of γδT cells.

[0016] The present invention also provides an application of a sea cucumber polysaccharide extract in improving the killing activity of γδT cells on lymphoma Daudi cells.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The gamma delta T cells obtained by the present invention have high killing activity against lymphoma Daudi cells. As the culture time increases, the killing activity increases to varying degrees, and the gamma delta T cells have high activity.

[0019] The present invention has high efficiency in amplifying γδT cells. The amplification multiple of γδT cells cultured in vitro for 10 days is greater than 2000 times, which is conducive to quickly obtaining highly active γδT cells and is simple and convenient to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a flow cytometry result diagram of the proportion of γδT cells cultured for 10 days in Example 4 of the present invention.

[0021] Figure 2 This is a graph showing the ratio of γδT cells obtained at different culture times according to the present invention.

[0022] Figure 3 The results of the γδT cell expansion times obtained at different culture times in the present invention are shown in FIG. DETAILED DESCRIPTION

[0023] In order to better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention.

[0024] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods.

[0025] Unless otherwise specified, the materials, reagents, etc. used in the examples of the present invention can be obtained from commercial sources.

[0026] Example 1 - Preparation of Sea Cucumber Polysaccharide Extract

[0027] Take 100g of sea cucumber powder, add 2L of 95% ethanol, mix, perform ultrasonic extraction at 60℃ and 600W for 60min, let it stand overnight, discard the supernatant, recover the ethanol, take precipitate 1; dissolve 500mL of deionized water in precipitate 1, add Sevag reagent (V 氯仿 :V 正丁醇 =4:1) 500mL was used for extraction to remove protein, the supernatant was collected, 95% ethanol 4 times the volume of the supernatant was added for precipitation, and the mixture was allowed to stand at 4°C overnight. The supernatant was discarded, the ethanol was recovered, and the mixture was centrifuged. Precipitate 2 was taken and freeze-dried to obtain Precipitate 2 powder; 5 g of Precipitate 2 powder was taken and dissolved in 50 mL of deionized water. The mixture was eluted through an HP-SAX strong anion exchange column with 0.4 M, 0.6 M, and 0.8 M NaCl solutions, respectively. The eluates at each level were collected, concentrated, dialyzed against tap water for 2 days, and freeze-dried to obtain a sea cucumber polysaccharide extract.

[0028] Example 2 - Preparation of Sea Cucumber Polysaccharide Extract

[0029] Take 100g of sea cucumber powder, add 2.5L of 95% ethanol, mix, perform ultrasonic extraction at 60℃ and 600W for 60min, let it stand overnight, discard the supernatant, recover the ethanol, take precipitate 1; dissolve 500mL of deionized water in precipitate 1, add Sevag reagent (V 氯仿 :V 正丁醇 =4:1) 500mL was used for extraction to remove protein, the supernatant was collected, 95% ethanol 5 times the volume of the supernatant was added for precipitation, and the mixture was allowed to stand at 4°C overnight. The supernatant was discarded, the ethanol was recovered, and the mixture was centrifuged. Precipitate 2 was taken and freeze-dried to obtain Precipitate 2 powder; 5 g of Precipitate 2 powder was taken and dissolved in 50mL of deionized water, and the mixture was passed through a DEAE-52 fiber column and eluted with 0.4M, 0.6M, and 0.8M NaCl solutions, respectively. The eluates at each level were collected, concentrated, dialyzed against tap water for 2d, and freeze-dried to obtain a sea cucumber polysaccharide extract.

[0030] Example 3 - Preparation of Sea Cucumber Polysaccharide Extract

[0031] Take 100g of sea cucumber powder, add 2L of 95% ethanol, mix, perform ultrasonic extraction at 60℃ and 600W for 60min, let it stand overnight, discard the supernatant, recover the ethanol, take precipitate 1; dissolve 500mL of deionized water in precipitate 1, add Sevag reagent (V 氯仿 :V 正丁醇 =4:1) 500mL was used for extraction to remove protein, the supernatant was collected, 95% ethanol 4 times the volume of the supernatant was added for precipitation, and the mixture was allowed to stand at 4°C overnight. The supernatant was discarded, the ethanol was recovered, and the mixture was centrifuged. Precipitate 2 was taken and freeze-dried to obtain Precipitate 2 powder; 5 g of Precipitate 2 powder was taken and dissolved in 50 mL of deionized water. The mixture was eluted through an HP-SAX strong anion exchange column with 0.4 M, 0.6 M, and 0.8 M NaCl solutions, respectively. The eluates at each level were collected, concentrated, dialyzed against tap water for 2 days, and freeze-dried to obtain a sea cucumber polysaccharide extract.

[0032] Example 4 - In vitro culture of highly active γδT cells

[0033] Peripheral blood was collected to separate PBMCs, and RPMI 1640 medium (containing 10% fetal bovine serum and 100M / mL IL-2) was added, vortexed, thoroughly mixed, and cultured in a 37°C incubator containing 5% CO2 and 95% O2. On the second day of in vitro culture, 38 μg / mL of the sea cucumber polysaccharide extract of Example 1 was added to the culture medium, and the cells were cultured in a 37°C incubator containing 20% ​​CO2 and 80% O2. On the fourth day of in vitro culture, the culture medium was replaced with new RPMI 1640 medium (containing 10% fetal bovine serum and 100M / mL IL-2), and 22 μg / mL of the sea cucumber polysaccharide extract was added to the culture medium. The cell density was adjusted to 4×10 5 / mL, place in a 37℃ incubator containing 10% CO2 and 90% O2, and continue to culture for 5 to 12 days to obtain highly active γδT cells.

[0034] Example 5 - In vitro culture of highly active γδT cells

[0035] Peripheral blood was collected to separate PBMCs, and RPMI 1640 medium (containing 10% fetal bovine serum and 100M / mL IL-2) was added. The cells were vortexed and thoroughly mixed, and then cultured in a 37°C incubator containing 5% CO2 and 95% O2. On the second day of in vitro culture, 30 μg / mL of the sea cucumber polysaccharide extract of Example 1 was added to the culture medium, and the cells were cultured in a 37°C incubator containing 5% CO2 and 95% O2. On the fourth day of in vitro culture, the culture medium was replaced with new RPMI 1640 medium (containing 10% fetal bovine serum and 100M / mL IL-2), and 30 μg / mL of the sea cucumber polysaccharide extract was added to the culture medium. The cell density was adjusted to 4×10 5 The cells were cultured in a 37°C incubator containing 5% CO2 and 95% O2 for 5 to 12 days to obtain highly active γδT cells.

[0036] Example 6 - In vitro culture of highly active γδT cells

[0037] Peripheral blood was collected to separate PBMCs, and RPMI 1640 medium (containing 10% fetal bovine serum, 100 M / mL IL-2, and 38 μg / mL of the sea cucumber polysaccharide extract of Example 1) was added. The cells were vortexed and thoroughly mixed, and then cultured in an incubator at 37°C containing 5% CO2 and 95% O2. On the 4th day of in vitro culture, the medium was replaced with new RPMI 1640 medium (containing 10% fetal bovine serum, 100 M / mL IL-2, and 22 μg / mL of the sea cucumber polysaccharide extract of Example 1), and the cell density was adjusted to 4 × 10 5 / mL, place in a 37℃ incubator containing 10% CO2 and 90% O2, and continue to culture for 5 to 12 days to obtain highly active γδT cells.

[0038] Example 7 - In vitro culture of highly active γδT cells

[0039] Peripheral blood was collected to separate PBMCs, and RPMI 1640 medium (containing 10% fetal bovine serum and 100M / mL IL-2) was added. The cells were vortexed and thoroughly mixed, and then cultured in a 37°C incubator containing 5% CO2 and 95% O2. On the second day of in vitro culture, 38 μg / mL of the sea cucumber polysaccharide extract of Example 2 was added to the culture medium, and the cells were cultured in a 37°C incubator containing 20% ​​CO2 and 80% O2. On the fourth day of in vitro culture, the culture medium was replaced with new RPMI 1640 medium (containing 10% fetal bovine serum and 100M / mL IL-2), and 22 μg / mL of the sea cucumber polysaccharide extract was added to the culture medium. The cell density was adjusted to 4×10 5 / mL, placed in a 37℃ incubator containing 10% CO2 and 90% O2, and cultured for 10 days to obtain highly active γδT cells.

[0040] Example 8 - In vitro culture of highly active γδT cells

[0041] Peripheral blood was collected to separate PBMCs, and RPMI 1640 medium (containing 10% fetal bovine serum and 100M / mL IL-2) was added. The cells were vortexed and thoroughly mixed, and then cultured in a 37°C incubator containing 5% CO2 and 95% O2. On the second day of in vitro culture, 38 μg / mL of the sea cucumber polysaccharide extract of Example 3 was added to the culture medium, and the cells were cultured in a 37°C incubator containing 20% ​​CO2 and 80% O2. On the fourth day of in vitro culture, the culture medium was replaced with new RPMI 1640 medium (containing 10% fetal bovine serum and 100M / mL IL-2), and 22 μg / mL of the sea cucumber polysaccharide extract was added to the culture medium. The cell density was adjusted to 4×10 5 / mL, placed in a 37℃ incubator containing 10% CO2 and 90% O2, and cultured for 10 days to obtain highly active γδT cells.

[0042] Comparative Example 1 - In vitro culture of γδT cells

[0043] PBMCs were isolated from peripheral blood and added to RPMI 1640 medium (containing 10% fetal bovine serum and 100 μg / mL IL-2). The cells were vortexed and thoroughly mixed, and then cultured in a 37°C incubator containing 5% CO2 and 95% O2. On the fourth day of in vitro culture, the medium was replaced with fresh RPMI 1640 medium (containing 10% fetal bovine serum and 100 μg / mL IL-2) and the cell density was adjusted to 4 × 10 5 The cells were cultured in a 37°C incubator containing 10% CO2 and 90% O2 for 10 to 12 days to obtain highly active γδT cells.

[0044] The amplification factor after 12 days was determined to be 1475 times.

[0045] Test Example 1

[0046] The number of γδT cells was calculated by cell counting method, and the γδT cells were detected by flow cytometry to calculate the expansion fold of γδT cells. Figure 1-3 It can be seen that the proportion of γδT cells is >90%, specifically 93.7%. At the same time, the expansion multiple is greater than 2000 times when cultured for 10 days, and specifically 2741 times in Example 4. The expansion efficiency is high, which is conducive to quickly obtaining highly active γδT cells. The expansion multiple of Example 5 is 2589 times when cultured for 10 days, indicating that the stimulation of sea cucumber polysaccharide extract and hypoxia is more conducive to the expansion of γδT cells. Example 6 has an expansion multiple of 2127 times when cultured for 12 days. The extracted sea cucumber polysaccharide has the function of improving cellular immunity, but premature addition to cell culture will cause cellular immunity and reduce the expansion multiple of γδT cells. Culturing sea cucumber polysaccharides with γδT cells can reduce the impact of the environment on the growth of γδT cells, protect cells from damage, and promote cytokines and stimulate the proliferation of γδT cells.

[0047] When γδT cells were cultured using the sea cucumber polysaccharide extract of Example 2 and the in vitro culture method of Example 4 (Example 7), the expansion factor was 2430 times after 10 days of culture; when γδT cells were cultured using the in vitro culture method of Example 4 of the sea cucumber polysaccharide extract of Example 3 (Example 8), the expansion factor was 2385 times after 10 days of culture.

[0048] Test Example 2

[0049] The cell line used was Daudi cell line (human Burkitt lymphoma cells). Daudi cells were resuscitated with warm water and inoculated into RPMI 1640 medium (containing 10% fetal bovine serum). The cells were cultured in a 37°C incubator containing 5% CO2 and 95% O2. Daudi cells in the logarithmic growth phase were collected. The cell density was adjusted to 1×10 5 Daudi cells (target cells) were seeded into 96-well plates, and γδT cells (effector cells) were added at an effector-target ratio of 20:1. The plates were cultured in a 37°C incubator containing 5% CO2 and 95% O2. The plates were centrifuged at 1500 rpm for 10 min, and the supernatant was collected. The OD value was recorded at 492 nm. The single effector cells and target cells were used as the control group to calculate the cell killing activity. Killing activity / % = (OD 样品 -OD 效应 ) / OD 靶 ×100, where OD 样品 is the measured OD value of the sample, OD 效应 is the OD value of effector cell release, OD 靶 is the maximum release value of target cells. The results are shown in Table 1.

[0050] Table 1 Cytotoxicity of γδT cells against Daudi cells

[0051] Time / h 12 24 48 72 Example 4 33.8±1.24 45.2±1.73 48.9±1.82 42.0±1.56 Comparative Example 1 21.3±0.95 28.7±1.11 27.0±1.08 25.7±1.03

[0052] Note: The γδT cells in Example 4 and Comparative Example 1 were all obtained by culturing for 10 days.

[0053] As shown in Table 1 above, at an effector-target ratio of 20:1, the cytotoxic activity of γδT cells against lymphoma Daudi cells was above 30% after co-culture for 12 h, 24 h, 48 h, and 72 h, respectively. The cytotoxic activity increased to varying degrees with prolonged culture time, with the effect being even better after 48 h. Furthermore, when the culture time was extended to 72 h, the cytotoxic activity of γδT cells against lymphoma Daudi cells still reached 40%, indicating that γδT cells have high activity.

[0054] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for culturing highly active γδT cells in vitro, characterized in that: The method comprises adding the sea cucumber polysaccharide extract to the culture medium on the 2nd to 3rd day of in vitro culture, and replacing the culture medium and adding the sea cucumber polysaccharide extract at the same time on the 4th to 5th day of in vitro culture; On the 2nd to 3rd day of in vitro culture, the amount of sea cucumber polysaccharide extract added was 30-40 μg / mL, and on the 4th to 5th day of in vitro culture, the amount of sea cucumber polysaccharide extract added was 20-30 μg / mL; When changing the culture medium, adjust the cell density to (4-5) × 10 5 / mL; On the second to third day of in vitro culture, the O2 volume concentration was adjusted to 75-80%, with the remainder being CO2. On the fourth to fifth day of in vitro culture, the O2 volume concentration was adjusted to 85-90%, with the remainder being CO2. The preparation method of the sea cucumber polysaccharide extract comprises the following steps: adding ethanol to sea cucumber powder, performing ultrasonic extraction, allowing the mixture to stand, discarding the supernatant, taking precipitate 1, adding Sevag reagent for extraction, collecting the supernatant, performing alcohol precipitation, discarding the supernatant, centrifuging, taking precipitate 2, freeze-drying, dissolving in water, passing through an HP-SAX strong anion exchange chromatography column, collecting the eluate, concentrating, dialysis, and freeze-drying to obtain the sea cucumber polysaccharide extract; The mass volume ratio of sea cucumber powder and ethanol is 1:20-25 mL; the Sevag reagent is chloroform and n-butanol in a volume ratio of 4:1; the HP-SAX strong anion exchange chromatography column is used for elution with 0.4 M, 0.6 M, and 0.8 M NaCl solutions, respectively.

2. A method for culturing highly active γδT cells in vitro according to claim 1, characterized in that: On the 2nd to 3rd day of in vitro culture, the added amount of sea cucumber polysaccharide extract was 38 μg / mL, and on the 4th to 5th day of in vitro culture, the added amount of sea cucumber polysaccharide extract was 22 μg / mL.

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