CN114517176A - Kit for inducing IPS (IPS) cells into NK (Natural killer) cells and application method thereof - Google Patents
Kit for inducing IPS (IPS) cells into NK (Natural killer) cells and application method thereof Download PDFInfo
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- CN114517176A CN114517176A CN202210100448.5A CN202210100448A CN114517176A CN 114517176 A CN114517176 A CN 114517176A CN 202210100448 A CN202210100448 A CN 202210100448A CN 114517176 A CN114517176 A CN 114517176A
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Abstract
The invention provides a kit for inducing IPS cells into NK cells, which comprises a first culture solution, a second culture solution and a third culture solution; the stem cell factor, the human bone morphogenetic protein and the vascular endothelial growth factor contained in the first culture solution can effectively induce the differentiation of mesoderm, and Y-27632 can obviously reduce the apoptosis of IPS cells. The components and other nutrient components in the culture medium are used in the process of forming the Spin EB by the IPS cells, and can promote the proliferation and differentiation of the Spin EB. The stem cell factor, interleukin 7, interleukin 15 and a ligand of a tyrosine kinase receptor 3 in the second culture solution and the third culture solution, and the interleukin 3 contained in the third culture solution can promote the early proliferation and differentiation of NK cells, and the components synergistically act on each stage of differentiation of Spin EB into NK cells, so that high-quantity and high-purity NK cells can be obtained.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a kit for inducing IPS cells into NK cells and an application method thereof.
Background
Induced pluripotent stem cells (IPS cells) can be differentiated in vitro into Embryoid Bodies (EBs) containing various germ layers under appropriate conditions, and further differentiated into target cell types, such as insulin-secreting cells, hematopoietic cells, neural cells, and the like. Since the IPS cell is directly reprogrammed by the somatic cell of the patient, immune rejection does not exist during cell transplantation, and the ethical problem of cell sources does not need to be considered, so that the IPS cell has wide clinical application prospect.
Natural killer cells, as a class of independent lymphocyte populations, can directly kill target cells, including tumor cells, virus or bacteria infected cells, and certain senescent normal cells of the body without antigen pre-sensitization, and are thus considered the first line of defense in the body's immune defense system. With the development of tumor immunology, more and more researches show that the application of natural killer cells (NK cells) to adoptive immunotherapy has obvious effects on eliminating residual tumor lesions and promoting the reconstruction of the immune system of a patient, and becomes an important therapeutic means for eliminating residual tumor cells after chemotherapy and hematopoietic stem cell transplantation. Clinical NK cells have the condition of insufficient supply, and currently, researches on inducing differentiation of NK cells include cell co-culture technology or methods of adding culture media and the like, but the methods have the defects of small cell number, low differentiation efficiency and susceptibility to pathogen pollution when the cells are successfully induced. Therefore, there is a need for further research and development of an optimal differentiation-inducing system capable of inducing IPS cells into NK cells.
Disclosure of Invention
In order to solve the above-mentioned problems, the present invention aims to provide a kit for inducing IPS cells into NK cells and use thereof, so that a culture induction system with high efficiency and stability and a simple and easy-to-operate culture method can be developed to obtain high-quality and high-purity NK cells.
According to a first aspect of the present invention, there is provided a kit for inducing IPS cells into NK cells, comprising the following components: a first culture solution, a second culture solution and a third culture solution;
the first culture solution includes: 1 part of cell basal medium, 1-10 mg/mL of recombinant human albumin, 1-10 mg/mL of deionized bovine serum albumin, 3-15 vol% of polyvinyl alcohol, 10-150 ng/mL of linoleic acid, 10-150 ng/mL of linolenic acid, 1-10 mug/mL of synthetic choline, 300-500 uM alpha-thioglycerol, 0.1-2 vol% of insulin-transferrin-selenium-ethanolamine solution, 2-10 vol% of protein-free hybridoma mixture II, 10-100 ug/mL of 2-phosphoric acid ascorbic acid, 5-150 ng/m of dry cell factor, 5-60 ng/mL of human bone morphogenetic protein, 5-60 ng/mL of vascular endothelial growth factor and 5-60 uMY-27632;
the second culture solution comprises: 2 parts of cell basal culture, 10-20 vol% of heat-inactivated human AB serum, 1-10 mM L-glutamine, 0.1-2 mu M beta-mercaptoethanol, 2-8 ng/mL of sodium selenite, 10-50 mu M of ethanolamine, 10-40 mg/L of ascorbic acid, 5-150 ng/mL of stem cell factor, 5-100 ng/mL of interleukin 7, 5-100 ng/mL of interleukin 15 and 1-50 ng/mL of tyrosine kinase receptor 3 ligand.
The third culture solution comprises: a second culture solution and 2-50 ng/mL interleukin 3.
The invention provides a kit for inducing IPS cells into NK cells and an application method thereof, wherein the first culture solution can culture the IPS cells to form single-cell aggregated embryoid bodies (Spin EBs), and the second culture solution and the third culture solution can induce the Spin EBs to be differentiated into the NK cells. The first culture solution does not contain serum or feeder cells, the content of the components of the culture medium is definite and controllable, exogenous pollution is not easy to cause, batch production is facilitated, the repeatability of experimental results is high, and the IPS cells can efficiently form Spin EBs. Through experimental research, the stem cell factor, the human bone morphogenetic protein and the vascular endothelial growth factor contained in the first culture solution can effectively induce the differentiation of mesoderm, and Y-27632 can obviously reduce the apoptosis of IPS cells and promote the proliferation of the IPS cells. The synergistic effect of the components can promote intercellular signal path conduction and other nutrient components in the culture solution, is used for forming the Spin EB by the IPS cells and contributes to the early proliferation and differentiation of the Spin EB into NK cells.
We found that the stem cell factor, interleukin 7, interleukin 15 and tyrosine kinase receptor 3 ligand (FLT-3L) in the second and third culture media act in combination on various stages of differentiation of Spin EB into NK cells. Stem cell factor and FLT-3L can promote differentiation of Spin EB, promote early development of NK cells and differentiate NK progenitor cells. The combined action of stem cell factor, interleukin 7, interleukin 15 and FLT-3L can promote the proliferation of Spin EB and further increase the number of NK cells. The interleukin 7 and the interleukin 15 can activate the NK cells, promote the rapid maturation differentiation, development and proliferation of the NK cells, improve the cytotoxic effect of the NK cells and promote the NK cells to secrete cytokines, thereby playing roles in immune regulation and killing. The interleukin 3 contained in the third culture solution can promote the proliferation and differentiation of NK cells in an early stage. The third culture solution and the second culture solution are applied to different stages of the differentiation of the Spin EB, the components have synergistic effect, and other nutrient components in the culture solution act on each stage of the differentiation from the Spin EB to the NK cells to the maturity, so that the differentiation efficiency is effectively improved, and the successful differentiation proportion of the NK cells is improved.
Preferably, the first culture solution comprises: 86.2 vol% of cell basal medium 1, 1-10 mg/mL of recombinant human albumin, 2-8 mg/mL of deionized bovine serum albumin, 5-10 vol% of polyvinyl alcohol, 10-100 ng/mL of linoleic acid, 10-100 ng/mL of linolenic acid, 2-8 mug/mL of synthetic choline, 350-450 uM alpha-thioglycerol, 0.1-1 vol% of insulin-transferrin-selenium-ethanolamine solution, 2-8 vol% of protein-free hybridoma mixture II, 10-100 ug/mL of 2-phosphoric acid ascorbic acid, 30-50 ng/m of dry cell factor, 10-30 ng/mL of human bone morphogenetic protein, 10-30 ng/mL of vascular endothelial growth factor and 5-15 uMY-27632;
the second culture solution comprises: 75-83 vol% cell basal culture 2, 10-20 vol% heat-inactivated human AB serum, 2mM L-glutamine, 0.8-1.2 mu M beta-mercaptoethanol, 2-8 ng/mL sodium selenite, 50 mu M ethanolamine, 10-40 mg/L ascorbic acid, 10-30 ng/mL stem cell factor, 10-30 ng/mL interleukin 7, 5-20 ng/mL interleukin 15 and 5-20 ng/mL tyrosine kinase receptor 3 ligand;
the third culture solution comprises: a second culture solution and 2-8 ng/mL interleukin 3.
By further limiting the content of each component in each culture medium, the kit provided by the invention has higher efficiency of inducing IPS cells into NK cells.
Preferably, the cell basal medium 1 consists of: IMDM medium: f12 medium 1:1, the first broth further comprising 2mm0.5 vol% penicillin-streptomycin.
Preferably, the cell basal medium 2 consists of: DMEM medium: and F12 culture medium is 5-5.5: 2.5-2.8, and the third culture solution further comprises 1 vol% of penicillin-streptomycin.
Preferably, the kit for inducing IPS cells into NK cells further comprises a fourth culture medium; the fourth culture solution was used during the centrifugation of IPS cells; the fourth culture solution comprises: 90 vol% cell basal medium 3 and 10 vol% fetal bovine serum.
Preferably, the cell basal medium 3 consists of the following components, calculated by volume ratio: DMEM medium: f12 medium 1: 1.
According to another aspect of the present invention, there is provided a method of inducing IPS cells into NK cells, comprising:
s1: culturing the IPS cells by adopting the first culture solution to form Spin EB;
s2: and inducing Spin EB into NK cells by using the second culture solution and the third culture solution.
Preferably, in S1, the specific steps are:
s1.1: inoculating the IPS cells into a 6-well plate paved with a Matrigel gel solution;
s1.2: when the IPS cells are cultured to grow to 70% -80% of fusion degree, washing the IPS cells with physiological saline for 2 times, and discarding washing liquid;
s1.3: adding preheated tryplE Select into an incubator for digestion for 3-10 min, blowing IPS cells into single cells, and transferring the single cells into a 15mL centrifuge tube;
s1.4: adding 5mL of fourth culture solution into the 15mL centrifuge tube, uniformly mixing, centrifuging, removing supernatant, and washing the IPS cells once with normal saline;
s1.5: resuspending the IPS cells by using a first culture solution, and adjusting the density of the IPS cells to obtain an IPS cell solution;
s1.6: and (3) inoculating the IPS cell liquid to a 96-hole round-bottom pore plate according to 100 mu L/hole, centrifuging, and transferring the 96-hole round-bottom pore plate to an incubator for culturing for 6 days to obtain the Spin EB liquid.
Preferably, the seeding density of the IPS cells in S1.1 is 10-50 ten thousand per hole; in S1.5, the density of IPS cells is adjusted to 5-20 ten thousand/mL.
In the step of culturing the IPS cells to form Spin EB, the IPS cells are digested into single cells by TryplE Select and then are further centrifuged to obtain a single cell centrifugal aggregation formed embryoid-like body (Spin EB). The number of IPS cells forming Spin EB affects the efficiency of further differentiation of EB cells, and when the number of cells is too small, the total number of NK cells successfully differentiated is low, and when the number of cells is too large, the efficiency of differentiation of EB cells is lowered.
Preferably, in S2, the specific steps are:
s2.1: adding 1-3% gelatin solution into a 6-hole plate according to 1 mL/hole, standing for 1h in an incubator to obtain a 6-hole plate paved with gelatin, and adding a third culture solution into the 6-hole plate;
s2.2: spin EB liquid was discarded most of the supernatant, which was added to the above 6-well plate
S2.3: after culturing for 6 days, changing into a second culture solution, and continuously culturing for 5-7 days;
s2.4: and continuously culturing for 3-4 weeks.
Preferably, in S2.4, the second culture solution is changed every 3 days from the 14 th day of culture.
The kit for inducing the IPS cells into the NK cells provided by the invention not only provides suitable nutritional conditions suitable for IPS cell differentiation and NK cell proliferation, but also can be added with various effective components capable of acting on various stages of IPS cell differentiation. By applying each culture solution in the kit to each step of induction and differentiation of IPS cell culture, NK cells with high differentiation efficiency, high purity and large quantity can be obtained.
Drawings
FIG. 1 is a microscope photograph of NK cells obtained by Spin EB-induced differentiation for 21 days in example 2.
Detailed Description
In order to make the technical solutions of the present invention better understood by those skilled in the art, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
Table 1 shows the content of the final components of the first culture solution, the second culture solution and the third culture solution in the kits provided in examples 1 to 3 and comparative examples 1 to 4. In addition, the kit provided by the embodiments 1-3 and the comparative examples 1-4 further comprises a fourth culture solution, and the kit comprises the following specific components: 90 vol% (DMEM medium: F12 medium 1:1) and 10 vol% fetal bovine serum.
TABLE 1 ingredient tables of first, second, and third culture solutions in examples 1 to 3 and comparative examples 1 to 4
Comparative example 5
The kit provided in the comparative example comprises a first culture solution, a second culture solution, a third culture solution and a fourth culture solution. The first culture solution comprises the following components: cell basal medium (IMDM: F12 ═ 1:1), 2mm 0.5% penicillin-streptomycin; the second culture solution and the third culture solution comprise the following components: 99 vol% cell basal medium (DEME: F12 ═ 2:1), 1 vol% penicillin-streptomycin; the fourth culture solution had a composition of 90 vol% (DMEM medium: F12 medium: 1) and 10 vol% fetal bovine serum.
The specific experimental operations for inducing IPS cells into NK cells using the kits provided in examples 1-3 and comparative examples 1-5 were as follows:
s1: culturing the IPS cells by adopting a first culture solution in the kit to form Spin EB;
s1.1: inoculating the IPS cells into a 6-well plate paved with a Matrigel glue solution according to the inoculation density of 50 ten thousand per well;
s1.2: when the IPS cells are cultured to grow to 80% fusion degree, washing the IPS cells with physiological saline for 2 times, and discarding washing liquid;
s1.3: adding preheated tryplE Select, digesting in an incubator for 6min, blowing IPS cells into single cells, and transferring the single cells into a 15mL centrifuge tube;
s1.4: adding 5mL of fourth culture solution into the 15mL centrifuge tube, uniformly mixing, centrifuging, removing supernatant, and washing the IPS cells once with normal saline;
s1.5: resuspending the IPS cells by using a first culture solution, and adjusting the density of the IPS cells to be 20 ten thousand/mL to obtain an IPS cell solution;
s1.6: and (3) inoculating the IPS cell liquid to a 96-hole round-bottom pore plate according to 100 mu L/hole, centrifuging, and transferring the 96-hole round-bottom pore plate to an incubator for culturing for 6 days to obtain the Spin EB liquid.
S2: inducing Spin EB into NK cells S2.1 by using a second culture solution and a third culture solution in the kit: adding a 3% gelatin solution into a 6-hole plate according to 1 mL/hole, standing in an incubator for 1h to obtain a 6-hole plate paved with gelatin, and adding a third culture solution into the 6-hole plate;
s2.2: removing most of supernatant of Spin EB liquid, and adding the Spin EB liquid into the 6-hole plate;
s2.3: changing into a second culture solution after culturing for 6 days, and continuously culturing for 5 days;
s2.4: the culture was continued for 3 weeks, and the second culture solution was changed every 3 days from the 14 th day of culture.
Test example
1. The subjects to be tested in this test example were NK cells in examples 1 to 3 and comparative examples 1 to 5.
CD3-Cell, CD56+The cells are the cellular immunophenotype mainly detected by the in vitro amplification culture of NK cells. The test example adopts a flow cytometry detection method: 5X 10 cell samples obtained at 7, 14 and 21 days after the induction culture of inducing Spin EB into NK cells in S2 were collected5Cells/tube, PBS wash 2 times. Adding flow detection antibodies respectively for double-labeled flow phenotype detection: FITC-labeled murine anti-human CD3 antibody and APC-labeled murine anti-human CD56 antibody; incubate at room temperature for 20min, wash 2 times with PBS, and analyze the cells by flow cytometry. The experimental results are shown in table 2; and counting the total number of cells, and the results are shown in table 3.
2. The cells induced to differentiate and cultured up to day 21 in S2.4 of example 2 were collected, and the morphology of the cells was observed by an optical microscope, as shown in FIG. 1. As can be seen from FIG. 1, in example 2, NK cell colonies were formed 21 days after the induction culture, and the number of formed NK cells was large and the cells were piled.
TABLE 2 CD3-Cell, CD56+Proportion of cells in the course of Induction culture
TABLE 3 Total cell count during induction culture
The experimental results show that the kit for inducing the IPS cells into the NK cells can smoothly culture the IPS cells to form the Spin EB, further can efficiently induce and differentiate the IPS cells into the NK cells, and has high differentiation efficiency, high cell number and high cell purity when the IPS cells are induced into the NK cells.
Although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that various changes, modifications and equivalents may be made therein without departing from the spirit and scope of the invention.
Claims (10)
1. A kit for inducing IPS cells into NK cells, comprising the following components: a first culture solution, a second culture solution and a third culture solution;
the first culture solution comprises: 1 part of cell basal medium, 1-10 mg/mL of recombinant human albumin, 1-10 mg/mL of deionized bovine serum albumin, 3-15 vol% of polyvinyl alcohol, 10-150 ng/mL of linoleic acid, 10-150 ng/mL of linolenic acid, 1-10 mug/mL of synthetic choline, 300-500 uM alpha-thioglycerol, 0.1-2 vol% of insulin-transferrin-selenium-ethanolamine solution, 2-10 vol% of protein-free hybridoma mixture II, 10-100 ug/mL of 2-phosphoric acid ascorbic acid, 5-150 ng/mL of dry cell factor, 5-60 ng/mL of human bone morphogenetic protein, 5-60 ng/mL of vascular endothelial growth factor and 5-60 uMY-27632;
the second culture solution comprises: 2 parts of cell basal medium, 10-20 vol% heat-inactivated human AB serum, 1-10 mML-glutamine, 0.1-2 MuM beta-mercaptoethanol, 2-8 ng/mL sodium selenite, 10-50 MuM ethanolamine, 10-40 mg/L ascorbic acid, 5-150 ng/mL stem cell factor, 5-100 ng/mL interleukin 7, 5-100 ng/mL interleukin 15 and 1-50 ng/mL tyrosine kinase receptor 3 ligand;
the third culture solution comprises: the second culture solution and 2-50 ng/mL interleukin 3.
2. The kit for inducing IPS cells into NK cells according to claim 1, wherein said cell basal medium 1 consists of, by volume: IMDM medium: f12 medium 1: 1; the first broth also included 2mm0.5 vol% penicillin-streptomycin.
3. The kit for inducing IPS cells into NK cells according to claim 1, wherein said cell basal medium 2 consists of, by volume: DMEM medium: and F12 culture medium is 5-5.5: 2.5 to 2.8; the second culture solution further comprises 1 vol% penicillin-streptomycin.
4. The kit for inducing IPS cells into NK cells of claim 1, further comprising a fourth culture fluid; the fourth culture solution comprises: cell basal medium 3 and 10 vol% fetal bovine serum.
5. The kit for inducing IPS cells into NK cells according to claim 4, wherein the cell basal medium 3 consists of the following components in volume ratio: DMEM medium: f12 medium 1: 1.
6. A method of inducing IPS cells into NK cells, comprising:
s1: culturing IPS cells to form Spin EBs using the first culture solution of claim 1;
s2: inducing said Spin EBs into NK cells using said second and third culture media of claim 1.
7. The method of inducing IPS cells as NK cells of claim 6, wherein in S1, the specific steps are:
s1.1: inoculating the IPS cells into a first culture vessel paved with a Matrigel gel solution;
s1.2: when the IPS cells are cultured to grow to 70% -80% of fusion degree, washing the IPS cells with normal saline, and discarding washing liquid;
s1.3: adding tryplE Select for digestion, blowing the IPS cells into single cells, and transferring the single cells into a centrifugal tube;
s1.4: adding a fourth culture solution into the centrifuge tube, uniformly mixing, centrifuging, removing supernatant, and washing the IPS cells with normal saline;
s1.5: resuspending the IPS cells by using the first culture solution, and adjusting the density of the IPS cells to obtain an IPS cell solution;
s1.6: and inoculating the IPS cell sap into a second culture vessel according to 100 mu L/hole, and culturing for 6 days after centrifugation to obtain the Spin EB liquid.
8. The method of inducing IPS cells as NK cells of claim 7, wherein the seeding density of the IPS cells in S1.1 is 10-50 ten thousand per well; in the S1.5, the density of the IPS cells is adjusted to be 5-20 ten thousand/mL.
9. The method of inducing IPS cells as NK cells of claim 8, wherein in S2, the specific steps are:
s2.1: adding the third culture solution into a third culture vessel paved with gelatin;
s2.2: removing supernatant of the Spin EB liquid, and adding the Spin EB liquid into the third culture vessel;
s2.3: after culturing for 6 days, changing the culture solution into the second culture solution, and continuously culturing for 5-7 days;
s2.4: and continuously culturing for 3-4 weeks.
10. The method of inducing IPS cells into NK cells of claim 9, wherein in S2.4, said second culture solution is changed every 3 days from the 14 th day of culture.
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| PCT/CN2023/071330 WO2023143006A1 (en) | 2022-01-27 | 2023-01-09 | Kit for inducing ips cells into nk cells and use method thereof |
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| WO2023143006A1 (en) * | 2022-01-27 | 2023-08-03 | 广东普罗凯融生物医药科技有限公司 | Kit for inducing ips cells into nk cells and use method thereof |
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