A novel in vitro culture method for human primary nk cell

Abstract

This invention discloses a novel method for culturing natural killer cells in vitro without added feeder cells. The method maintains and expands natural killer cells isolated from umbilical cord blood of normal healthy volunteers using a culture medium composition. This culture medium composition comprises culture medium, heat-inactivated fetal bovine serum, antibiotics (penicillin-streptomycin), and two cytokines, IL-2 and IL-15. This culture medium composition provides a safe and functional material and method for culturing natural killer cells.

Description

Technical Field

[0001] This invention relates to a culture medium composition and culture method for in vitro expanded NK cells. Prior Technology

[0002] In recent years, in addition to traditional surgery, chemotherapy and radiation therapy, there has been another option for cancer treatment: immunotherapy.

[0003] Cell therapy involves administering a specific type of cell to a patient to treat their disease. The cell manufacturing process affects the effective quantity, safety, and efficacy of the cells, and each batch of cells produced by each cell processing unit (CPU) varies in quantity, quality, safety, and efficacy.

[0004] Natural killer cells (NK cells) are a type of non-specific lymphocytes that possess cytotoxic activity but lack T-cell and B-cell receptors. NK cells are key members of the innate immune system, capable of directly killing target cells (infected cells, tumor cells) without prior exposure to antigens. Their killing effect is not restricted by the major histocompatibility complex (MHC), and they possess anti-infective, anti-tumor, and immunomodulatory functions.

[0005] Natural killer cell therapy is divided into three categories: autologous, allogeneic (including umbilical cord blood), and cell line cultured. Natural killer cells are isolated from the patient's blood, expanded and activated in vitro, and then reinfused into the body. The resulting therapy is primarily composed of natural killer cells. Natural killer cells can recognize and kill cancerous cells in the body without specific antigen stimulation. Furthermore, natural killer cells can indirectly activate the immune system by secreting cytokines, thereby enhancing anti-cancer capabilities. However, the required quantity of natural killer cells to induce an effective immunotoxic effect and the quality of the prepared natural killer cells to achieve an effective immunotoxic effect remain challenges.

[0006] In the past, when implementing in vitro cell expansion and activation methods, natural killer cells and feeder cells were often co-cultured to significantly increase the number of natural killer cells. However, these cells would quickly lose their function in vivo. Furthermore, there are safety concerns with expansion methods that use cancer cells as feeder cells. Before infusing the expanded natural killer cells into the patient, it is difficult to determine whether the cancer feeder cells have been completely removed, thus raising safety concerns.

[0007] Therefore, it is necessary to develop a method for in vitro culture and expansion of natural killer cells without the use of trophoblast cells, so as to obtain safe natural killer cells that can maintain their function in vivo. Summary of the Invention

[0008] In view of this, the inventor of this case has a deep understanding of the shortcomings and defects of the previous case, and has been eager to improve and innovate it. After years of research, the inventor has successfully developed a culture medium composition and culture method for in vitro culture and expansion of human natural killer cells.

[0009] To achieve the above objectives, the present invention provides a culture medium composition comprising a basal culture medium, heat-inactivated fetal bovine serum, antibiotics, and cytokines, wherein the cytokines are IL-2 and IL-15.

[0010] In one embodiment of the present invention, the basal culture medium is a stem cell growth medium.

[0011] In one embodiment of the present invention, the antibiotic comprises penicillin and streptomycin.

[0012] In one embodiment of the present invention, the amount of IL-2 present in the culture medium composition is 0.01~1 U / mL, and the amount of IL-15 present in the culture medium composition is 0.001~0.1 U / mL.

[0013] In one embodiment of the present invention, the heat-inactivated fetal bovine serum is present in the culture medium composition at an amount of 4-6% (v / v).

[0014] In one embodiment of the present invention, the amount of the antibiotic present in the culture medium composition is 0.8-1.2% (v / v).

[0015] The present invention also provides a method for culturing natural killer cells, comprising the following steps: Step a: Mononuclear cells (MNCs) isolated from human umbilical cord blood; Step b: Isolate natural killer cells from these monocytes; Step c: The natural killer cells are cultured in the aforementioned culture medium composition; Step d: More than 80% of the resulting cell population consists of natural killer cells, which are CD3-CD56+ cells. The number of natural killer cells in step d is more than 1.5 times that in step b.

[0016] In one embodiment of the present invention, the characteristic feature is that step b is to obtain natural killer cells using a human natural killer cell isolation kit.

[0017] In one embodiment of the present invention, no trophoblast cells are added in the method.

[0018] This invention provides a novel in vitro culture medium composition for culturing and expanding natural killer cells without the addition of feeder cells, and provides a safe and functional natural killer cell culture medium composition and culture method thereof. Simple Explanation of the Diagram

[0019] Figure 1 shows the cell density scaling results of the natural killer cells of this invention.

[0020] Figure 2A shows the phenotypic analysis results of the natural killer cells of the present invention after staining with CD3-CD56+ antibody.

[0021] Figure 2B shows the purity results of the natural killer cells of this invention after staining with CD3-CD56+ antibody. Implementation

[0022] The words "or," "and," and "and" used in this specification, unless otherwise stated, refer to "or / and." Furthermore, the terms "comprising" and "including" are not restrictive open-ended conjunctions. The foregoing paragraphs are for systematic reference only and should not be construed as limiting the subject of the invention.

[0023] Unless otherwise specified, "%" in this manual refers to "volume percentage (V%)"; numerical ranges (e.g., 10%~11% of A) include upper and lower limits unless otherwise specified (i.e., 10%≦A≦11%); if no lower limit is defined for a numerical range (e.g., less than 0.2% of B, or B below 0.2%), then the lower limit may be 0 (i.e., 0%≦B≦0.2%).

[0024] All values ​​disclosed in this specification are subject to a standard technical measurement error (standard deviation) of ±10%. The term "about" is intended to indicate ±10%, ±5%, ±2.5%, or ±1% relative to a given value; that is, "about 20%" represents 20 ±2%, 20 ±1%, 20 ±0.5%, or 20 ±0.25%.

[0025] As used in this specification, the terms "enhance," "boost," "enhance," or similar terms refer to increasing or prolonging the intended efficacy or duration of the culture medium composition. Therefore, "enhance" in the context of enhancing the stimulatory and activating efficacy of the culture medium composition refers to an increased or prolonged capacity in other therapeutic systems, which is a capacity for efficacy or duration.

[0026] The term "synergistic effect" as used in this specification means that the sum of the effects of individual factors or active agents acting simultaneously is greater than the effect of any single factor.

[0027] In one embodiment, the compositions are administered in the form of solutions, suspensions, emulsions, powders, slurries, colloids, or topical agents.

[0028] The present invention is illustrated by the following embodiments, but the present invention is not limited to the following embodiments.

[0029] [Novel In Vitro Culture Method for Natural Killer Cells: Culture Compositions and Their Applications]

[0030] This invention provides a culture composition for a novel in vitro culture method of natural killer cells, comprising a basal culture medium and heat-inactivated fetal bovine serum (FBS). The content of FBS depends on the type of FBS. In one embodiment, the content of FBS is approximately 4-6% (v / v), for example, approximately 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, or any two of the above values. In one embodiment, it is preferably approximately 4-6% (v / v). By keeping the content of FBS within the above range, its component compatibility, stimulation and activation, and cell uptake can be balanced, exhibiting a synergistic effect and further enhancing the proliferation of natural killer cells in the culture medium composition.

[0031] This invention provides a culture composition for a novel in vitro culture method of natural killer cells, comprising a basal culture medium and an antibiotic, wherein the antibiotic content depends on the type of antibiotic; in one embodiment, the antibiotic content is about 0.5-1.5% (v / v), for example, about 0.10%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.80%, 0.85%, 0.90%, 0.95%, 1.00%, 1.05%, 1.10%, 1.15%, 1.20%, 1.25%, 1.30%, 1.35%, 1.40%, 1.45%, 1.50%, 1.55%, 1.60%, 1.65%, 1.70%. The content is %, 1.75%, 1.80%, 1.85%, 1.90%, 1.95%, 2.00%, or any two of the above values; in one embodiment, it is preferably about 0.5~1.5% (v / v); by keeping the content of the antibiotic within the above range, its component compatibility, stimulation activation and cell absorption can be taken into account, and a synergistic effect can be presented, which can further enhance the proliferation of natural killer cells by the culture medium composition.

[0032] The present invention provides a culture composition for a novel in vitro culture method for natural killer cells, which comprises a basal medium and cytokines, wherein the content of the cytokines is determined according to the types of the cytokines; in one embodiment, the content of IL-2 is about 0.01~1 U / mL, for example, can be about 0.01 U / mL, 0.02 U / mL, 0.03 U / mL, 0.04 U / mL, 0.05 U / mL, 0.06 U / mL, 0.07 U / mL, 0.08 U / mL, 0.09 U / mL, 0.10 U / mL, 0.11 U / mL, 0.12 U / mL, 0.13 U / mL, 0.14 U / mL, 0.15 U / mL, 0.16 U / mL, 0.17 U / mL, 0.18 U / mL, 0.19 U / mL, 0.20 U / mL, 0.21 U / mL, 0.22 U / mL, 0.23 U / mL, 0.24 U / mL, 0.25 U / mL, 0.26 U / mL, 0.27 U / mL, 0.28 U / mL, 0.29 U / mL, 0.30 U / mL, 0.31 U / mL, 0.32 U / mL, 0.33 U / mL, 0.34 U / mL, 0.35 U / mL, 0.36 U / mL, 0.37 U / mL, 0.38 U / mL, 0.39 U / mL, 0.40 U / mL, 0.41 U / mL, 0.42 U / mL, 0.43 U / mL, 0.44 U / mL, 0.45 U / mL, 0.46 U / mL, 0.47 U / mL, 0.48 U / mL, 0.49 U / mL, 0.50 U / mL, 0.51 U / mL, 0.52 U / mL, 0.53 U / mL, 0.54 U / mL, 0.55 U / mL, 0.56 U / mL, 0.57 U / mL, 0.58 U / mL, 0.59 U / mL, 0.60 U / mL, 0.61 U / mL, 0.62 U / mL, 0.63 U / mL, 0.64 U / mL, 0.65 U / mL, 0.66 U / mL, 0.67 U / mL, 0.68 U / mL, 0.69 U / mL, 0.70 U / mL, 0.71 U / mL, 0.72 U / mL, 0.73 U / mL, 0.74 U / mL, 0.75 U / mL, 0.76 U / mL, 0.77 U / mL, 0.78 U / mL, 0.79 U / mL, 0.80 U / mL, 0.81 U / mL, 0.82 U / mL, 0.83 U / mL, 0.84 U / mL, 0.85 U / mL, 0.86 U / mL, 0.87 U / mL, 0.88 U / mL, 0.89 U / mL, 0.The concentrations are 90 U / mL, 0.91 U / mL, 0.92 U / mL, 0.93 U / mL, 0.94 U / mL, 0.95 U / mL, 0.96 U / mL, 0.97 U / mL, 0.98 U / mL, 0.99 U / mL, 1 U / mL, or any two of the above values; in one embodiment, it is preferably about 0.01~1 U / mL. By keeping the IL-2 concentration within the above range, its compatibility, stimulatory activation, and cell uptake can be balanced, exhibiting a synergistic effect and further enhancing the proliferation of natural killer cells in the culture medium composition.

[0033] This invention provides a culture composition for a novel in vitro culture method of natural killer cells, comprising a basal culture medium and cytokines, wherein the content of the cytokines depends on the type of cytokines; in one embodiment, the content of IL-15 is about 0.001~0.1 U / mL, for example, about 0.001 U / mL, 0.002 U / mL, 0.003 U / mL, 0.004 U / mL, 0.005 U / mL, 0.006 U / mL, 0.007 U / mL, 0.008 U / mL, 0.009 U / mL, 0.010 U / mL, 0.011 U / mL, 0.012 U / mL, 0.013 U / mL, 0.014 U / mL, 0.015 U / mL, 0.016 U / mL, 0.017 U / mL, 0.018 U / mL, 0.019 U / mL, 0.020 U / mL, etc. U / mL, 0.021 U / mL, 0.022 U / mL, 0.023 U / mL, 0.024 U / mL, 0.025 U / mL, 0.026 U / mL, 0.027 U / mL, 0.028 U / mL, 0.029 U / mL, 0.030 U / mL, 0.031 U / mL, 0.032 U / mL, 0.033 U / mL, 0.034 U / mL, 0.035 U / mL, 0.036 U / mL, 0.037 U / mL, 0.009 U / mL, 0.009 U / mL, 0.009 U / mL, 0.009 U / mL, 0.009 U / mL, 0.009 U / mL, 0.038 U / mL, 0.039 U / mL, 0.040 U / mL, 0.041 U / mL, 0.042 U / mL, 0.043 U / mL, 0.044 U / mL, 0.045 U / mL, 0.046 U / mL, 0.047 U / mL, 0.048 U / mL, 0.049 U / mL, 0.050 U / mL, 0.051 U / mL, 0.052 U / mL, 0.053 U / mL, 0.054 U / mL, 0.055 U / mL, 0.056 U / mL, 0.057 U / mL, 0.058 U / mL, 0.059 U / mL, 0.060 U / mL, 0.061 U / mL, 0.062 U / mL, 0.063 U / mL, 0.064 U / mL, 0.065 U / mL, 0.066 U / mL, 0.067 U / mL, 0.068 U / mL, 0.069 U / mL, 0.070 U / mL, 0.071 U / mL, 0.072 U / mL, 0.073 U / mL, 0.074 U / mL, 0.075 U / mL, 0.076 U / mL, 0.077 U / mL, 0.078 U / mL, 0.079 U / mL, 0.080 U / mL, 0.081 U / mL, 0.082 U / mL, 0.083 U / mL, 0.084 U / mL, 0.085 U / mL, 0.086 U / mL, 0.087 U / mL, 0.088 U / mL, 0.089 U / mL, 0.090 U / mL, 0.091 U / mL, 0.092 U / mL, 0.093 U / mL, 0.094 U / mL, 0.095 U / mL, 0.096 U / mL, 0.097 U / mL, 0.098 U / mL, 0.099 U / mL, 0.1 U / mL, or the content between any two of the above values; in one embodiment, it is preferably about 0.01~0.1 U / mL; by maintaining the IL-15 content within the above range, it is possible to balance component compatibility, stimulation and activation, and cell uptake, exhibiting a synergistic effect and further enhancing the proliferation of natural killer cells in the culture medium composition.

[0034] This invention provides a novel in vitro culture method for natural killer cells. The purity of the natural killer cells is determined by staining with CD3-CD56+ antibodies. In one embodiment, the purity index is approximately 75-100%, for example, it can be approximately 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, 99%, 99%, 99%, 100%, or any two of the above values. In one embodiment, the lower limit is approximately 80%; the higher limit is approximately 95%. By ensuring that the purity index is within the above range, it can be confirmed that the compatibility of the culture composition increases the stimulation and activation of natural killer cells and enhances cell uptake, thereby exhibiting a synergistic effect to improve the proliferation and quality of natural killer cells.

[0035] This invention provides a novel in vitro culture method for natural killer cells, and the culture composition is used for preparing pharmaceutical compositions, wherein the use is for the effect of rapid proliferation of natural killer cells; in one embodiment, cell density ratio and antibody staining are used to identify purity; cell density and purity identification can be selected according to the expression form or the desired effect, but the invention is not limited thereto.

[0036] The following examples provide comparative illustrations of the compositions of the present invention, but are not intended to limit the invention. Any person skilled in the art can make various modifications and refinements without departing from the spirit and scope of the invention. The above and other objects, features, and advantages of the present invention will become clearer from the following description and the accompanying drawings.

[0037] [Preparation Example] [1] [Cultivation composition and preparation process]

[0038] As shown in Table 1, the culture medium composition has 5 components that need to be prepared and mixed before use: 1. Prepare a 1-fold dilution of the stem cell growth medium. Based on a total volume of 50 mL for the entire mixture, the required volume of stem cell growth medium is 47 mL ± 3 µL / mL. 2. The volume of heat-inactivated fetal bovine serum diluted 100% to 5% is calculated to be 2.5 mL ± 2 µL / mL, relative to the total volume of the mixture (50 mL). 3. The antibiotic (penicillin-streptomycin) is diluted 100 times to a 1-fold dilution, with the total volume of the combination being 50 mL. The amount of antibiotic used in this dilution is 500 µL ± 2 µL / mL. 4. Dilute 100 U / mL of cytokine IL-2 to 0.1 U / mL. Based on a total volume of 50 mL for the entire mixture, the amount of IL-2 used in this dilution is 50 µL. 5. Dilute 100 U / mL of cytokine IL-15 to 0.01 U / mL. The amount of IL-2 used in this dilution is 50 µL, which is calculated relative to the total volume of the combination of IL-15 and IL-15.

[0039] [surface] [1] [Composition and Formulation of a Novel In Vitro Culture Method]

[0040] [Efficacy Test Example] [1] [Natural killer cell density analysis]

[0041] The cell density of the culture composition of the novel in vitro culture method for natural killer cells of the present invention was tested after culture; the cultured natural killer cells were read in a cell counter, and the growth rate after culture was confirmed by using day 0 as the denominator.

[0042] Referring to Figure 1, higher cell density leads to better proliferation of natural killer cells in the culture medium. Cell density was calculated on days 0, 1, 3, 5, 7, 10, and 12. Because no feeder cells were present, only two cytokines were added, and the dosage was lower than in known systems. Therefore, cell density decreased on days 1 and 3, but a significant proliferation effect began on day 5, reaching a 1.5-fold increase by day 12. This result shows that the culture medium composition resulted in approximately 7 times higher proliferation of natural killer cells on day 12 compared to day 3.

[0043] [Efficacy Test Example] [1] [Natural Killer Cell Purity Analysis]

[0044] The cell purity of the culture composition of the novel in vitro culture method for natural killer cells of the present invention was tested after culture; the cultured natural killer cells were stained with CD3-CD56+ antibody and then read by flow cytometry to confirm the cell purity of the antibody staining.

[0045] Please refer to Figure 2A. For confirming cell health morphology, forward scattered light (FSC) can analyze cell size, and scattered light (SSC) can analyze cell granularity. These characteristics can be used to distinguish whether cells are healthy. Cells undergoing apoptosis will have smaller FSC values, while SSC values ​​will first increase and then decrease. This principle can be used to qualitatively characterize cells. Figure 2A shows that the circled area represents 88.2% of natural killer cells with good cell health and also stained with CD3-CD56+ antibodies. The purity percentage of these 88.2% healthy cells can be obtained from Figure 2B. The higher the antibody staining level, the higher the purity, which will appear in the lower right quadrant of Figure 2B. The results in Figure 2B show that the purity of natural killer cells maintained and expanded using a culture medium composition without feeder cells is 96.1%. This means that when 10,000 cells are read by flow cytometry, 96.1% of the cells are natural killer cells stained with CD3-CD56+ antibodies.

[0046] In summary, this invention provides a novel in vitro culture medium composition in which cytokines contain only low doses of IL-2 and IL-15. Natural killer cells are cultured and expanded without the addition of feeder cells. Cell density analysis shows that the feeder-free culture medium composition resulted in approximately 7 times higher concentrations of natural killer cells on day 12 compared to day 3. Flow cytometry analysis shows that the purity of natural killer cells maintained and expanded using the feeder-free culture medium composition is 96.1%. These experimental results demonstrate that this invention provides a feeder-free culture medium composition that effectively maintains and expands natural killer cells with high purity, providing a safe and functional culture medium composition for natural killer cells and its culture method.

[0047] none

Claims

1. A culture medium composition comprising a basal medium, 5-20% heat-inactivated fetal bovine serum, 1-10% antibiotics, and 0.001-1% cytokines, the cytokines being IL-2 and IL-15.

2. The culture medium composition as claimed in claim 1, wherein the basal culture medium is a stem cell growth medium.

3. The culture medium composition as claimed in claim 1, wherein the antibiotic comprises penicillin and streptomycin.

4. The culture medium composition as claimed in claim 1, wherein the amount of IL-2 present in the culture medium composition is 0.01 to 1 U / mL, and the amount of IL-15 present in the culture medium composition is 0.001 to 0.1 U / mL.

5. The culture medium composition as claimed in claim 1, wherein the heat-inactivated fetal bovine serum is present in the culture medium composition at an amount of 4 to 6% (v / v).

6. The culture medium composition as claimed in claim 1, wherein the antibiotic is present in the culture medium composition at an amount of 0.5 to 1.5% (v / v).

7. A method for culturing natural killer cells, comprising the following steps: Step a: isolating monocytes (MNCs) from human umbilical cord blood; Step b: isolating natural killer cells from the monocytes; Step c: culturing the natural killer cells in a culture medium composition as described in claim 1; Step d: obtaining a cell population in which more than 80% are natural killer cells, the natural killer cells being CD3-CD56+ cells, wherein the amount of natural killer cells in step d is more than 1.5 times that in step b.

8. The culture method as described in claim 7, characterized in that step b is to obtain natural killer cells using a human natural killer cell isolation kit.

9. The method as described in claim 7, wherein no trophoblast cells are added.

10. The culture method described in claim 7, wherein more than 95% of the cell population obtained in step d are natural killer cells.

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