Culture medium and method for differentiation of stem cells into macrophages

Abstract

The application provides a culture medium and method for stem cell differentiation into macrophages, and relates to the technical field of biotechnology.The culture medium provided by the application comprises six kinds of culture media, the components of which are clear, and the culture media and cytokines are prepared without animal sources, so that exogenous substances are avoided, the differentiated cells are safer, and clinical transformation can be seamlessly connected.The preparation of macrophages by using the culture medium does not need to pass through an EB process, so that the steps are simplified, the differentiation time is shortened, the purity, uniformity and stability of differentiation are improved.The method for differentiating macrophages provided by the application skips the complicated blastocyst formation stage, simplifies the differentiation steps, greatly shortens the differentiation period, reduces unknown risks, improves the differentiation efficiency and stability, can obtain a large amount of high-purity macrophages with killing capacity in a short period, and also provides the possibility of full-closed automatic production for large-scale production preparation, and solves the bottleneck of industrialization difficulty.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a culture medium and method for differentiating stem cells into macrophages. Background Technology

[0002] Tumor immunotherapy, especially adoptive immunotherapy, has developed rapidly in recent years. Chimeric antigen receptor (CAR) modified T cells have shown good efficacy in the treatment of hematological malignancies, but they still have certain limitations in the treatment of solid tumors. Macrophages (Macs) are important immune cells in the body, possessing phagocytic and antigen-presenting functions, and participating in anti-tumor, anti-viral infection, and immune regulation processes.

[0003] Macrophages exhibit cytotoxic effects against various types of tumors and can be used in combination with other immunotherapies. In particular, chimeric antigen receptor-modified macrophages (CAR-Macs) have been used in the treatment of malignant solid tumors in recent years, demonstrating strong tumor-killing capabilities and significantly prolonging overall survival. Currently, there are two methods for obtaining Macs for clinical treatment. One is to extract and differentiate Macs (P-Macs) from peripheral blood mononuclear cells (PBMCs) of patients and genetically modify them through adenovirus infection. This method has the advantages of not causing immune rejection and a short production cycle, but it has disadvantages such as extremely high cost, difficulty in controlling product quality (highly dependent on the patient's condition), limited cell production quantity, difficulty in gene editing, and lack of universality. The other method is to differentiate Macs from induced pluripotent stem cells (iPSCs) (iMacs). This method effectively addresses the shortcomings of P-Macs, while iPSCs have unlimited expansion capabilities, are easy to modify, and can be gene-edited. Existing differentiation methods require the formation of embryoid bodies (EBs), followed by centrifugation to create cell clumps that aggregate in well plates. EB formation necessitates culturing in a specific differentiation medium for 7-10 days before collection and transfer to other well plates for further differentiation, a process that requires trypsin treatment. While EB formation is beneficial for stem cell differentiation, it is labor-intensive and time-consuming. Furthermore, the use of different additives in EB formation results in cell products at varying stages, compromising product homogeneity and posing challenges to large-scale and stable production. Therefore, a simple, stable, serum-free culture medium and EB-free intermediate culture and differentiation process are needed to enable fully closed-loop industrial-scale differentiation. However, current iMac differentiation methods are cumbersome, time-consuming, unstable, and involve unclear or animal-derived culture medium compositions, hindering industrial-scale production. Therefore, a simple, stable differentiation method that can rapidly produce large quantities of clinically needed Macs is essential.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] The first objective of this invention is to provide a set of culture media for the differentiation of stem cells into macrophages, in order to solve at least one of the above-mentioned problems.

[0006] A second objective of this invention is to provide an application of the above-mentioned culture medium in the differentiation and culture of pluripotent stem cells to obtain macrophages.

[0007] A third objective of this invention is to provide a method for differentiating macrophages.

[0008] In a first aspect, the present invention provides a set of culture media for the differentiation of stem cells into macrophages, the culture media comprising a first culture medium, a second culture medium, a third culture medium, a fourth culture medium, a fifth culture medium, and a sixth culture medium;

[0009] The first culture medium includes Essential 8 medium, mTeSR plus medium, STEMdiff APEL2 medium, or CTS. TM AIM-V TM SFM medium, CHIR990212-10μM, BMP4 50-250ng / mL and VEGF 50-250ng / mL;

[0010] The second culture medium includes Essential 6 medium, mTeSR plus medium, STEMdiff APEL2 medium, or CTS. TM AIM-V TM SFM medium, SB 2-20μM, VEGF 30-250ng / mL, SCF 30-200ng / mL and bFGF 30-200ng / mL;

[0011] The third culture medium includes ImmunoCult-SF Macrophage Medium, MethoCult H4034 Optimum Medium, StemSpan XF Medium, StemPro-34 Medium, Stemline II Medium, BIT 9500 Serum Substitute Medium, MethoCult H4330 Medium, MyeloCult H5100 Medium, or CTS AIM-V SFM Medium, with VEGF 10-250 ng / mL, SCF 30-200 ng / mL, TPO 2-50 ng / mL, IL3 20-250 ng / mL, FLT3L 20-200 ng / mL, and bFGF 20-250 ng / mL.

[0012] The fourth culture medium includes ImmunoCult-SF Macrophage Medium, StemSpan XF medium, Stemline II medium, MethoCult H4034 Optimum medium, BIT 9500 Serum Substitute medium, MethoCult H4330 medium, MyeloCult H5100 medium, or CTS AIM-V SFM medium, SCF 20-200 ng / mL, TPO 2-50 ng / mL, IL3 50-250 ng / mL, FLT3L 20-200 ng / mL, and M-CSF 20-200 ng / mL;

[0013] The fifth culture medium includes ImmunoCult-SF Macrophage Medium, X-VIVO 15, StemSpan XF, Stemline II, MethoCult H4034 Optimum, BIT9500 Serum Substitute, MethoCult H4330, MyeloCult H5100, CTSAIM-V SFM, FLT3L 30-200 ng / mL, GM-CSF 20-100 ng / mL, and M-CSF 30-200 ng / mL;

[0014] The sixth culture medium includes ImmunoCult-SF Macrophage Medium, X-VIVO 15, StemSpan XF, Stemline II, MethoCult H4034 Optimum, BIT9500 Serum Substitute, MethoCult H4330, MyeloCult H5100, CTSAIM-V SFM, GM-CSF 30-200 ng / mL, and M-CSF 30-200 ng / mL.

[0015] As a further technical solution, the first culture medium includes Essential 8 medium, STEMdiff APEL2 medium, mTeSR plus medium, or CTS medium. TM AIM-V TM SFM medium, CHIR99021 4μM, BMP4 80ng / mL and VEGF 80ng / mL;

[0016] The second culture medium includes Essential 6 medium, STEMdiff APEL 2 medium, mTeSRplus medium, or CTS. TM AIM-V TM SFM medium, SB 2μM, VEGF 40ng / mL, SCF 50ng / mL and bFGF 50ng / mL;

[0017] The third culture medium includes ImmunoCult-SF Macrophage Medium, StemSpan XF, StemPro-34, Stemline II, MethoCult H4034 Optimum, BIT9500 Serum Substitute, MethoCult H4330, MyeloCult H5100, or CTSAIM-V SFM, with VEGF 40 ng / mL, SCF 50 ng / mL, TPO 10 ng / mL, IL3 50 ng / mL, FLT3L 50 ng / mL, and bFGF 50 ng / mL.

[0018] The fourth culture medium includes ImmunoCult-SF Macrophage Medium, StemSpan XF medium, Stemline II medium, MethoCult H4034 Optimum medium, BIT 9500 Serum Substitute medium, MethoCult H4330 medium, MyeloCult H5100 medium, or CTS AIM-V SFM medium, SCF 50 ng / mL, TPO 10 ng / mL, IL3 50 ng / mL, FLT3L 50 ng / mL, and M-CSF 50 ng / mL;

[0019] The fifth culture medium includes ImmunoCult-SF Macrophage Medium, X-VIVO 15, StemSpan XF, Stemline II, MethoCult H4034 Optimum, BIT9500 Serum Substitute, MethoCult H4330, MyeloCult H5100, CTSAIM-V SFM, FLT3L 50 ng / mL, GM-CSF 25 ng / mL, and M-CSF 50 ng / mL;

[0020] The sixth culture medium includes ImmunoCult-SF Macrophage Medium, MethoCult H4034 Optimum Medium, X-VIVO 15 Medium, StemSpan XF Medium, Stemline II Medium, BIT 9500 Serum Substitute Medium, MethoCult H4330 Medium, MyeloCult H5100 Medium, CTS AIM-V SFM Medium, GM-CSF 50 ng / mL, and M-CSF 50 ng / mL.

[0021] Secondly, the present invention provides the application of the above-mentioned culture medium in differentiating and culturing pluripotent stem cells to obtain macrophages.

[0022] As a further technical solution, the pluripotent stem cells include induced pluripotent stem cells.

[0023] Thirdly, the present invention provides a method for differentiating macrophages, comprising the following steps: culturing pluripotent stem cells sequentially using the first to sixth culture media described above to differentiate them into macrophages.

[0024] a. Seed pluripotent stem cells into a culture vessel and culture until the pluripotent stem cells adhere to the vessel.

[0025] b. After the pluripotent stem cells adhere to the culture medium, change the culture medium to the first culture medium, change the culture medium to the second culture medium on the 3rd-4th day, change the culture medium to the third culture medium on the 5th-6th day, change the culture medium to the fourth culture medium on the 7th-8th day, and change the culture medium to the fifth culture medium on the 10th-11th day.

[0026] c. After obtaining monocytes through culture, culture the monocytes in the sixth medium for 2-3 days to obtain mature macrophages.

[0027] As a further technical solution, the pluripotent stem cells include induced pluripotent stem cells.

[0028] As a further technical solution, the pluripotent stem cells are gene-edited pluripotent stem cells.

[0029] As a further technical solution, the culture container is coated with Vitronectin, Laminin 521, or iMatrix-511.

[0030] As a further technical solution, the concentration of the pluripotent stem cells seeded is 0.5 × 10⁻⁶. 3 / cm 2 -5×10 3 / cm 2 .

[0031] As a further technical solution, the concentration of the mononuclear cells seeded into the sixth culture medium is 0.5 × 10⁻⁶. 3 / cm 2 -5×10 3 / cm 2 .

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The culture medium provided by this invention has a clearly defined composition and is prepared using animal-free medium and cytokines, avoiding the introduction of exogenous substances. This results in safer differentiated cells that can be seamlessly integrated into clinical translation. Differentiation of macrophages using this medium eliminates the need for the EB process, simplifying the steps, shortening the differentiation time, and improving the purity, uniformity, and stability of the differentiated cells.

[0034] The method for differentiating macrophages provided by this invention skips the cumbersome embryoid formation stage, simplifies the differentiation steps, greatly shortens the differentiation cycle, reduces unknown risks, and improves differentiation efficiency and stability. It can obtain a large number of high-purity macrophages with killing ability in a short period of time, and also provides the possibility of fully enclosed automated production for large-scale production, solving the bottleneck of industrialization difficulties. Attached Figure Description

[0035] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0036] Figure 1 Schematic diagrams of the Fast-iMac process for Example 1 and its comparison group, and Example 2;

[0037] Figure 2 Cell morphology diagrams of various stages of Fast-iMac in Example 1;

[0038] Figure 3 Flow cytometry results of differentiated cells on day 7 in Example 1 and its control group, and Example 2;

[0039] Figure 4 Flow cytometry results of differentiated cells on day 13 in Example 1 and its control group, and Example 2;

[0040] Figure 5Flow cytometry results of differentiated cells on day 15 in Example 1 and its control group, and Example 2;

[0041] Figure 6 Flow cytometry results of differentiated cells on day 15 in Example 1 and its control group, and Example 2, showing CD80, CD86, and CD163 levels.

[0042] Figure 7 Phagocytic results of mature iMac tumors in Examples 1 and 2;

[0043] Figure 8 Figures showing the results of tumor killing dynamics detection in differentiated mature iMac tumors in Examples 1 and 2;

[0044] Figure 9 Amplification curves of the iMac differentiated in Example 1 and its control group, and Example 2;

[0045] Figure 10 Fold-up plots of iMacs differentiated in Example 1 and its control group, and Example 2, on day 31. Detailed Implementation

[0046] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0047] The inventors discovered that current immunotherapy methods involve a long overall cycle for obtaining immune cells. Even expanding the required immune cells from autologous blood takes more than three weeks, and obtaining the desired functional cells through gene editing requires at least a month. Traditional differentiation methods are time-consuming, use culture media and matrix gels with unclear compositions or containing animal-derived components, and yield small cell numbers. Obtaining the required number of immune cells often requires significant human, material, and financial resources, greatly increasing the time and cost of immunotherapy. This invention provides a culture medium and method for obtaining large quantities of iMac cells in a short period, and the obtained iMac cells exhibit significant killing effects, which is of great significance for the immunotherapy of tumor cells.

[0048] In a first aspect, the present invention provides a set of culture media for the differentiation of stem cells into macrophages, the culture media comprising a first culture medium, a second culture medium, a third culture medium, a fourth culture medium, a fifth culture medium, and a sixth culture medium;

[0049] The first culture medium includes Essential 8 medium, mTeSR plus medium, STEMdiff APEL2 medium, or CTS. TM AIM-V TM SFM medium, CHIR99021, BMP4 and VEGF, wherein the concentration of CHIR99021 can be, for example, but not limited to, 2μM, 4μM, 6μM, 8μM or 10μM; the concentration of BMP4 can be, for example, but not limited to, 50ng / mL, 100ng / mL, 150ng / mL, 200ng / mL or 250ng / mL; the concentration of VEGF can be, for example, but not limited to, 50ng / mL, 100ng / mL, 150ng / mL, 200ng / mL or 250ng / mL.

[0050] The second culture medium includes Essential 6 medium, mTeSR plus medium, STEMdiff APEL2 medium, or CTS. TM AIM-V TM SFM medium, SB, VEGF, SCF, and bFGF. The concentration of SB can be, for example, but not limited to, 2 μM, 5 μM, 10 μM, 15 μM, or 20 μM; the concentration of VEGF can be, for example, but not limited to, 30 ng / mL, 50 ng / mL, 100 ng / mL, 150 ng / mL, 200 ng / mL, or 250 ng / mL; the concentration of SCF can be, for example, but not limited to, 30 ng / mL, 50 ng / mL, 100 ng / mL, 150 ng / mL, or 200 ng / mL; and the concentration of bFGF can be, for example, but not limited to, 30 ng / mL, 50 ng / mL, 100 ng / mL, 150 ng / mL, or 200 ng / mL.

[0051] The third culture medium includes ImmunoCult-SF Macrophage Medium, MethoCult H4034 Optimum Medium, StemSpan XF Medium, StemPro-34 Medium, Stemline II Medium, BIT 9500 Serum Substitute Medium, MethoCult H4330 Medium, MyeloCult H5100 Medium, or CTS AIM-V SFM Medium, VEGF, SCF, TPO, IL3, FLT3L, and bFGF. The concentrations of VEGF can be, for example, but not limited to, 10 ng / mL, 50 ng / mL, 100 ng / mL, 150 ng / mL, 200 ng / mL, or 250 ng / mL; the concentrations of SCF can be, for example, but not limited to, 30 ng / mL, 50 ng / mL, 100 ng / mL, 150 ng / mL, or 200 ng / mL; the concentrations of TPO can be, for example, but not limited to, 2 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, or 50 ng / mL; IL The concentration of 3 can be, for example, but not limited to, 20 ng / mL, 50 ng / mL, 100 ng / mL, 150 ng / mL, 200 ng / mL or 250 ng / mL; the concentration of FLT3L can be, for example, but not limited to, 20 ng / mL, 50 ng / mL, 100 ng / mL, 150 ng / mL or 200 ng / mL; the concentration of bFGF can be, for example, but not limited to, 20 ng / mL, 50 ng / mL, 100 ng / mL, 150 ng / mL, 200 ng / mL or 250 ng / mL.

[0052] The fourth culture medium includes ImmunoCult-SF Macrophage Medium, StemSpan XF Medium, Stemline II Medium, MethoCult H4034 Optimum Medium, BIT 9500 Serum Substitute Medium, MethoCult H4330 Medium, MyeloCult H5100 Medium, CTS AIM-V SFM Medium, SCF, TPO, IL3, FLT3L, and M-CSF. The concentrations of SCF can be, for example, but not limited to, 20 ng / mL, 50 ng / mL, 100 ng / mL, 150 ng / mL, or 200 ng / mL; the concentrations of TPO can be, for example, but not limited to, 2 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, or 50 ng / mL; the concentrations of IL3 can be, for example, but not limited to, 50 ng / mL, 100 ng / mL, 150 ng / mL, 200 ng / mL, or 250 ng / mL; the concentrations of FLT3L can be, for example, but not limited to, 20 ng / mL, 50 ng / mL, 100 ng / mL, 150 ng / mL, or 200 ng / mL; and the concentrations of M-CSF can be, for example, but not limited to, 20 ng / mL, 50 ng / mL, 100 ng / mL, 150 ng / mL, or 200 ng / mL.

[0053] The fifth culture medium includes ImmunoCult-SF Macrophage Medium, X-VIVO 15, StemSpan XF, Stemline II, MethoCult H4034 Optimum, BIT9500 Serum Substitute, MethoCult H4330, MyeloCult H5100, CTSAIM-V SFM, GM-CSF, FLT3L, and M-CSF. The concentration of FLT3L can be, for example, but not limited to, 30 ng / mL, 50 ng / mL, 100 ng / mL, 150 ng / mL or 200 ng / mL; the concentration of GM-CSF can be, for example, but not limited to, 20 ng / mL, 40 ng / mL, 60 ng / mL, 80 ng / mL or 100 ng / mL; and the concentration of M-CSF can be, for example, but not limited to, 30 ng / mL, 50 ng / mL, 100 ng / mL, 150 ng / mL or 200 ng / mL.

[0054] The sixth culture medium includes ImmunoCult-SF Macrophage Medium, X-VIVO 15 Medium, StemSpan XF Medium, Stemline II Medium, MethoCult H4034 Optimum Medium, BIT9500 Serum Substitute Medium, MethoCult H4330 Medium, MyeloCult H5100 Medium, CTSAIM-V SFM Medium, GM-CSF, and M-CSF. The concentration of GM-CSF may be, for example, but not limited to, 30 ng / mL, 50 ng / mL, 100 ng / mL, 150 ng / mL, or 200 ng / mL; the concentration of M-CSF may be, for example, but not limited to, 30 ng / mL, 50 ng / mL, 100 ng / mL, 150 ng / mL, or 200 ng / mL.

[0055] The culture medium provided by this invention has a clearly defined composition and is prepared using animal-free medium and cytokines, avoiding the introduction of exogenous substances. This results in safer differentiated cells that can be seamlessly integrated into clinical translation. Differentiation of macrophages using this medium eliminates the need for the EB process, simplifying the steps, shortening the differentiation time, and improving the purity, uniformity, and stability of the differentiated cells.

[0056] In some preferred embodiments, the first culture medium includes Essential 8 medium, STEMdiffAPEL 2 medium, mTeSR plus medium, or CTS. TM AIM-V TM SFM medium, CHIR99021 4μM, BMP4 80ng / mL and VEGF 80ng / mL;

[0057] The second culture medium includes Essential 6 medium, STEMdiff APEL 2 medium, mTeSRplus medium, or CTS. TM AIM-V TM SFM medium, SB 2μM, VEGF 40ng / mL, SCF 50ng / mL and bFGF 50ng / mL;

[0058] The third culture medium includes ImmunoCult-SF Macrophage Medium, StemSpan XF, StemPro-34, Stemline II, MethoCult H4034 Optimum, BIT 9500 Serum Substitute, MethoCult H4330, MyeloCult H5100, or CTS AIM-V SFM, with VEGF 40 ng / mL, SCF 50 ng / mL, TPO 10 ng / mL, IL3 50 ng / mL, FLT3L 50 ng / mL, and bFGF 50 ng / mL.

[0059] The fourth culture medium includes ImmunoCult-SF Macrophage Medium, StemSpan XF medium, Stemline II medium, MethoCult H4034 Optimum medium, BIT 9500 Serum Substitute medium, MethoCult H4330 medium, MyeloCult H5100 medium, or CTS AIM-V SFM medium, SCF 50 ng / mL, TPO 10 ng / mL, IL3 50 ng / mL, FLT3L 50 ng / mL, and M-CSF 50 ng / mL;

[0060] The fifth culture medium includes ImmunoCult-SF Macrophage Medium, X-VIVO 15, StemSpan XF, Stemline II, MethoCult H4034 Optimum, BIT9500 Serum Substitute, MethoCult H4330, MyeloCult H5100, CTSAIM-V SFM, FLT3L 50 ng / mL, GM-CSF 25 ng / mL, and M-CSF 50 ng / mL;

[0061] The sixth culture medium includes ImmunoCult-SF Macrophage Medium, MethoCult H4034 Optimum Medium, X-VIVO 15 Medium, StemSpan XF Medium, Stemline II Medium, BIT 9500 Serum Substitute Medium, MethoCult H4330 Medium, MyeloCult H5100 Medium, CTS AIM-V SFM Medium, GM-CSF 50 ng / mL, and M-CSF 50 ng / mL.

[0062] Further optimization and adjustment of the concentration of culture medium components resulted in higher culture efficiency, enabling the production of a large number of highly pure macrophages with cytotoxic capabilities in a short time.

[0063] Secondly, the present invention provides the application of the above-mentioned culture medium in differentiating and culturing pluripotent stem cells to obtain macrophages.

[0064] The culture medium provided by this invention has a clearly defined composition and is prepared using animal-free culture medium and cytokines, avoiding the introduction of exogenous substances. The differentiated cells are safer and can be used to differentiate pluripotent stem cells into macrophages.

[0065] In some preferred embodiments, the pluripotent stem cells include induced pluripotent stem cells (iPSCs).

[0066] Thirdly, the present invention provides a method for differentiating macrophages, comprising the following steps: culturing pluripotent stem cells sequentially using the first to sixth culture media described above to differentiate them into macrophages.

[0067] a. Seed pluripotent stem cells into a culture vessel and culture until the pluripotent stem cells adhere to the vessel.

[0068] b. After the pluripotent stem cells adhere to the culture medium, change the culture medium to the first culture medium, change the culture medium to the second culture medium on the 3rd-4th day, change the culture medium to the third culture medium on the 5th-6th day, change the culture medium to the fourth culture medium on the 7th-8th day, and change the culture medium to the fifth culture medium on the 10th-11th day.

[0069] c. After obtaining monocytes through culture, culture the monocytes in the sixth medium for 2-3 days to obtain mature macrophages.

[0070] The cell differentiation method described above significantly shortens the time to obtain iMacs, reducing it to two weeks and yielding a large number of cells. Monocytes can also be continuously collected for differentiation. This method can obtain a large number of iMacs with high purity in a short period of time.

[0071] In some preferred embodiments, the pluripotent stem cells include human induced pluripotent stem cells.

[0072] In some preferred embodiments, pluripotent stem cells can also be genetically modified, including but not limited to the insertion of chimeric antigen receptor genes, the insertion of polarization elements, the insertion of homing elements, and the knockout of immune checkpoints.

[0073] In some preferred embodiments, the culture container includes a petri dish or a culture plate.

[0074] In some preferred embodiments, the culture container is coated with Vitronectin (VTN), Laminin 521 (LN521), or iMatrix-511.

[0075] Using animal-free VTN, LN521, or iMatrix-511 as the cell matrix avoids the use of other cell lines as feeder layers, enabling differentiated Macs to meet clinical standards, making them safer and reducing unknown risks.

[0076] In some preferred embodiments, the VTN coating concentration is 50 ng / ml, and the diluent used is DPBS.

[0077] In some preferred embodiments, the concentration of the pluripotent stem cells seeded is 0.5 × 10⁻⁶. 3 / cm 2 -5×10 3 / cm 2 Preferably 1×10 3 / cm 2 .

[0078] In some preferred embodiments, the concentration of the mononuclear cells seeded into the sixth culture medium is 0.5 × 10⁻⁶. 3 / cm 2 -5×10 3 / cm 2 Preferably 1×10 3 / cm 2 .

[0079] The present invention will be further illustrated below with specific embodiments and comparative examples. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0080] Example 1

[0081] The training process is as follows Figure 1 As shown ( Figure 1The differentiation media 1a, 1b, 2a, 2b, 3, and 4 in this embodiment are, in order, the first, second, third, fourth, fifth, and sixth culture media. The cell morphology at each stage of culture is as follows: Figure 2 As shown.

[0082] 1) Dilute VTN to 50 ng / ml with DPBS, add 1 mL to each well of a six-well plate, and incubate at room temperature for 3 hours.

[0083] 2) Select healthy human induced pluripotent stem cells (HCPs) at appropriate densities for digestion. Digest with ReLeSR digestion solution for 5 min, then add an equal volume of DPBS to terminate the digestion. Centrifuge at 300g for 5 min. Resuspend in mTeSR plus solution. Discard the supernatant in the coated six-well plate. Add 2 mL of the resuspended HCPs to each well of the six-well plate, at a seeding concentration of 1×10⁻⁶. 4 .

[0084] 3) After 12 hours, replace mTeSR plus with the first culture medium and record it as day D0.

[0085] 4) On day 3, replace the first culture medium with the second culture medium.

[0086] 5) On day 5, replace the second culture medium with the third culture medium.

[0087] 6) On day 7, replace the third culture medium with the fourth culture medium.

[0088] 7) On day 10, replace the fourth culture medium with the fifth culture medium.

[0089] 8) On day 13, collect the Monocytes from the supernatant, centrifuge at 300g for 5 minutes, resuspend in medium VI, and add 1×10⁻⁶ mol / L. 5 / cm 2 Differentiation was carried out by inoculating the seeds into six-well plates.

[0090] 9) A fully-fledged iMac can be obtained in D15 days.

[0091] 10) In this protocol, the first culture medium is formulated as follows: STEMdiff APEL 2 with 4 μM CHIR99021, 80 ng / mL BMP4, and 80 ng / mL VEGF; the second culture medium is formulated as follows: STEMdiff APEL 2 with 2 μM SB, 40 ng / mL VEGF, 50 ng / mL SCF, and 50 ng / mL bFGF; the third culture medium is formulated as follows: StemSpan XF with 40 ng / mL VEGF, 50 ng / mL SCF, 10 ng / mL TPO, 50 ng / mL IL3, 50 ng / mL FLT3L, and 50 ng / mL bFGF; the fourth culture medium is formulated as follows: StemSpan XF ... XF medium contains 50 ng / mL SCF, 10 ng / mL TPO, 50 ng / mL IL3, 50 ng / mL FLT3L, and 50 ng / mL M-CSF. The fifth medium is X-VIVO 15 medium containing 50 ng / mL FLT3L, 25 ng / mL GM-CSF, and 50 ng / mL M-CSF. The sixth medium is X-VIVO 15 medium containing 50 ng / mL GM-CSF and 50 ng / mL M-CSF.

[0092] control group

[0093] For comparison, a control group was also set up in this embodiment. The only difference between the control group and this embodiment is that the concentration of cytokines was halved.

[0094] Example 2

[0095] This embodiment is a variation of Example 1, the only difference being the use of pluripotent stem cells expressing the chimeric antigen receptor gene for differentiation. The chimeric antigen receptor contains antibody sequences or receptor / ligand sequences that specifically recognize tumor cell surface antigens, as well as an intracellular domain. Through specific recognition of tumor cell surface antigens and activation of intracellular signaling pathways, it specifically phagocytoses and kills tumor cells.

[0096] Example 3

[0097] The difference from Example 1 lies in the culture medium. Specifically, the first culture medium consisted of mTeSRplus with 2 μM CHIR99021, 250 ng / mL BMP4, and 50 ng / mL VEGF; the second culture medium consisted of mTeSRplus with 2 μM SB, 250 ng / mL VEGF, 30 ng / mL SCF, and 200 ng / mL bFGF; the third culture medium consisted of StemPro-34 with 10 ng / mL VEGF, 200 ng / mL SCF, 2 ng / mL TPO, 250 ng / mL IL3, 20 ng / mL FLT3L, and 250 ng / mL bFGF; and the fourth culture medium consisted of StemPro-34 with... The first medium consisted of StemSpan XF containing 20 ng / mL SCF, 50 ng / mL TPO, 50 ng / mL IL3, 220 ng / mL FLT3L, and 20 ng / mL M-CSF. The second medium consisted of StemSpan XF containing 30 ng / mL FLT3L, 100 ng / mL GM-CSF, and 200 ng / mL M-CSF. The third medium consisted of StemSpan XF containing 30 ng / mL GM-CSF and 200 ng / mL M-CSF.

[0098] Example 4

[0099] The difference from Example 1 lies in the culture medium. Specifically, the first culture medium consisted of Essential 8 with 10 μM CHIR99021, 50 ng / mL BMP4, and 250 ng / mL VEGF; the second culture medium consisted of Essential 6 with 20 μM SB, 50 ng / mL VEGF, 30 ng / mL SCF, and 30 ng / mL bFGF; the third culture medium consisted of Stemline II with 250 ng / mL VEGF, 30 ng / mL SCF, 50 ng / mL TPO, 20 ng / mL IL3, 200 ng / mL FLT3L, and 20 ng / mL bFGF; and the fourth culture medium consisted of Stemline II with 200 ng / mL VEGF, 50 ng / mL BMP4, and 200 ng / mL bFGF. The fifth culture medium consisted of Stemline II with the addition of 200 ng / mL SCF, 20 ng / mL TPO, 250 ng / mL IL3, 20 ng / mL FLT3L, and 200 ng / mL M-CSF. The sixth culture medium consisted of Stemline II with the addition of 200 ng / mL GM-CSF and 30 ng / mL M-CSF.

[0100] Results of tests in Examples 1, 2, 3, 4 and the control group

[0101] Flow cytometry results testing:

[0102] Flow cytometry detection of CD34, CD45, CD11b, CD80, CD86, and CD14: Cells obtained from the examples and control groups were collected into 50 mL centrifuge tubes and centrifuged at 500 g for 5 min. After discarding the supernatant, the cells were resuspended in 1 mL of DPBS containing 2% BSA. The corresponding antibodies were added, and the cells were incubated at 4°C for 30 min. The cells were then washed once with DPBS containing 2% BSA. The cells were resuspended in an appropriate amount of DPBS to a density of 1 × 10⁻⁶ cells / mL. 6 / mL, the results were detected and analyzed using a flow cytometer.

[0103] In vitro tumor phagocytic capacity test:

[0104] 1) HepG2 cells stably expressing red fluorescent RFP were digested with Trypsin-EDTA, centrifuged at 300g for 5 min, resuspended in DMEM containing 10% FBS, and counted. Cells were then analyzed at 1×10⁻⁶ cells / mL. 4 / well inoculated into a 24-well plate;

[0105] 2) Accutase digested differentiated and matured iMac cells stably expressing green fluorescent GFP, centrifuged at 300g for 5 min, resuspended the cell pellet in DMEM containing 10% FBS and counted the cells, then added 1×10⁻⁶ cells to a 24-well plate seeded with tumor cells. 5 / well of iMac cells, and add culture medium to 1 mL / well;

[0106] 3) Gently place the 24-well plate into IncuCyte and take red and green fluorescence photos every 4 hours.

[0107] In vitro lethality test:

[0108] 4) HepG2 cells stably expressing red fluorescent RFP were digested with Trypsin-EDTA, centrifuged at 300g for 5 min, resuspended in DMEM containing 10% FBS, and counted. Cells were then analyzed at a concentration of 1×10⁻⁶ cells / mL. 4 / well inoculated into 24-well plates, with three replicates for each treatment group;

[0109] 5) Digest differentiated and mature iMac cells with Accutase, centrifuge at 300g for 5 min, resuspend the cell pellet in DMEM containing 10% FBS and count the cells. Add 1×10⁻⁶ cells to a 24-well plate inoculated with tumor cells. 5 / well of iMac cells, and add culture medium to 1 mL / well;

[0110] 6) Gently place the 24-well plate into the IncuCyte and take a picture every 2 hours.

[0111] Cell expansion fold detection:

[0112] The number of monocytes collected each day during the 30-day differentiation period was collected and recorded. The fold increase of differentiation was obtained by dividing the total number of monocytes collected by the number of initial iPSCs.

[0113] result:

[0114] The flow cytometry results of the hematopoietic stem cell marker CD34 are as follows: Figure 3 As shown: Flow cytometry analysis revealed that the CD34 positivity rate in Example 1 was 46% (compared to 29% in the control group), while the CD34 positivity rate in Example 2 was 38.9%; the flow cytometry results for the Monocyte marker CD14 are as follows. Figure 4 As shown: the CD14 positivity rate in Example 1 was 75.9% (compared to 47.3% in the control group), and the CD14 positivity rate in Example 2 was 74%; the flow cytometry results for the Mac marker CD11b are as follows. Figure 5As shown: the CD11b positivity rate in Example 1 was 90.9% (compared to 83.5% in the control group), and the CD11b positivity rate in Example 2 was 89.5%; the flow cytometry results of the Mac polarization markers are as follows. Figure 6 As shown: In Example 1, the positive rates of M1 polarization marker CD80 were 7.38% (compared to 18.79% in the control group), CD86 were 95.3% (compared to 88.4% in the control group), and the positive rate of M2 polarization marker CD163 was 38.2% (compared to 28.3% in the control group); In Example 2, the positive rates of M1 polarization marker CD80 were 8.87%, CD86 were 88.7%, and the positive rate of M2 polarization marker CD163 was 26.3%.

[0115] Examples 3 and 4 represent endpoint tests of factor concentrations in this differentiation protocol. The results showed that the positive rates for CD34 in Examples 3 and 4 were both below 20%; the positive rates for CD14 were both below 50%; the positive rates for CD11b and CD86 were both below 50%; the positive rate for CD80 was both below 5%; and the positive rate for CD163 was both below 10%. However, even at the endpoints of the differentiation factor concentrations, this protocol could still differentiate Macs, although the purity and fold change were not high, it still possessed the ability to differentiate Macs. Given that the purity and quantity of Macs differentiated in Examples 3 and 4 were not high, functional identification of the differentiated Macs was not performed in this study.

[0116] The results of tumor phagocytic capacity tests in Examples 1 and 2 are as follows: Figure 7 As shown, both Example 1 and Example 2 exhibit phagocytic activity against tumor cells. Compared to Mac derived from wild-type pluripotent stem cells, iMac expressing the chimeric antigen receptor gene exhibits stronger phagocytic activity against tumor cells and a longer duration of activity.

[0117] The lethality test results of Examples 1 and 2 are as follows: Figure 8 As shown, the killing effects of Examples 1 and 2 are significant. Compared with wild-type pluripotent stem cell-derived Mac, iMac expressing chimeric antigen receptor gene has a stronger killing ability against tumor cells.

[0118] The amplification fold test results of Example 1, its control group, and Example 2 are as follows: Figure 9 and Figure 10 As shown, the amplification folds of Examples 1 and 2 both reached the level of 100,000 times.

[0119] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A culture medium for differentiating stem cells into macrophages, characterized in that, The culture medium includes a first culture medium, a second culture medium, a third culture medium, a fourth culture medium, a fifth culture medium, and a sixth culture medium; The first culture medium is Essential 8 medium, mTeSR plus medium, STEMdiff APEL 2 medium, or CTS. TM AIM-V TM SFM medium, CHIR99021 2-10μM, BMP4 50-250ng / mL and VEGF 50-250ng / mL; The second culture medium is Essential 6 medium, mTeSR plus medium, STEMdiff APEL 2 medium, or CTS. TM AIM-V TM SFM medium, SB 2-20μM, VEGF 30-250ng / mL, SCF 30-200ng / mL and bFGF 30-200ng / mL; The third culture medium is ImmunoCult-SF Macrophage Medium, MethoCult H4034 Optimum Medium, StemSpan XF Medium, StemPro-34 Medium, Stemline II Medium, BIT 9500 Serum Substitute Medium, MethoCult H4330 Medium, MyeloCult H5100 Medium, or CTS AIM-V SFM Medium, containing VEGF 10-250 ng / mL, SCF 30-200 ng / mL, TPO 2-50 ng / mL, IL3 20-250 ng / mL, FLT3L 20-200 ng / mL, and bFGF 20-250 ng / mL. The fourth culture medium is ImmunoCult-SF Macrophage Medium, StemSpan XF medium, Stemline II medium, MethoCult H4034 Optimum medium, BIT 9500 Serum Substitute medium, MethoCult H4330 medium, MyeloCult H5100 medium, or CTS AIM-VSFM medium, with SCF 20-200 ng / mL, TPO 2-50 ng / mL, IL3 50-250 ng / mL, FLT3L 20-200 ng / mL, and M-CSF 20-200 ng / mL. The fifth culture medium is ImmunoCult-SF Macrophage Medium, X-VIVO 15, StemSpan XF, Stemline II, MethoCult H4034 Optimum, BIT9500 Serum Substitute, MethoCult H4330, MyeloCult H5100, CTSAIM-V SFM, FLT3L 30-200 ng / mL, GM-CSF 20-100 ng / mL, and M-CSF 30-200 ng / mL. The sixth culture medium is ImmunoCult-SF Macrophage Medium, X-VIVO 15, StemSpan XF, Stemline II, MethoCult H4034 Optimum, BIT9500 Serum Substitute, MethoCult H4330, MyeloCult H5100, CTSAIM-V SFM, GM-CSF 30-200 ng / mL, and M-CSF 30-200 ng / mL.

2. The culture medium according to claim 1, characterized in that, The first culture medium is Essential 8 medium, STEMdiffAPEL 2 medium, mTeSR plus medium, or CTS. TM AIM-V TM SFM medium, CHIR99021 4μM, BMP4 80ng / mL and VEGF 80ng / mL; The second culture medium is Essential 6 medium, STEMdiff APEL 2 medium, mTeSR plus medium, or CTS. TM AIM-V TM SFM medium, SB 2μM, VEGF 40ng / mL, SCF 50ng / mL and bFGF 50ng / mL; The third culture medium is ImmunoCult-SF Macrophage Medium, StemSpan XF medium, StemPro-34 medium, Stemline II medium, MethoCult H4034 Optimum medium, BIT9500 Serum Substitute medium, MethoCult H4330 medium, MyeloCult H5100 medium, or CTSAIM-V SFM medium, with VEGF 40 ng / mL, SCF 50 ng / mL, TPO 10 ng / mL, IL3 50 ng / mL, FLT3L 50 ng / mL, and bFGF 50 ng / mL. The fourth culture medium is ImmunoCult-SF Macrophage Medium, StemSpan XF medium, Stemline II medium, MethoCult H4034 Optimum medium, BIT 9500 Serum Substitute medium, MethoCult H4330 medium, MyeloCult H5100 medium, or CTS AIM-V SFM medium, SCF 50 ng / mL, TPO 10 ng / mL, IL3 50 ng / mL, FLT3L 50 ng / mL, and M-CSF 50 ng / mL; The fifth culture medium is ImmunoCult-SF Macrophage Medium, X-VIVO 15, StemSpan XF, Stemline II, MethoCult H4034 Optimum, BIT9500 Serum Substitute, MethoCult H4330, MyeloCult H5100, CTSAIM-V SFM, FLT3L 50 ng / mL, GM-CSF 25 ng / mL, and M-CSF 50 ng / mL. The sixth culture medium is ImmunoCult-SF Macrophage Medium, MethoCult H4034 Optimum Medium, X-VIVO 15 Medium, StemSpan XF Medium, Stemline II Medium, BIT9500 Serum Substitute Medium, MethoCult H4330 Medium, MyeloCult H5100 Medium, CTSAIM-V SFM Medium, GM-CSF 50 ng / mL, and M-CSF 50 ng / mL.

3. The application of the culture medium according to claim 1 or 2 in the differentiation and culture of pluripotent stem cells to obtain macrophages.

4. The application according to claim 3, characterized in that, The pluripotent stem cells include induced pluripotent stem cells.

5. A method for differentiating macrophages, characterized in that, Pluripotent stem cells are cultured sequentially using the first to sixth culture media as described in claim 1 or 2 to differentiate them into macrophages, comprising the following steps: a. Seed pluripotent stem cells into a culture vessel and culture until the pluripotent stem cells adhere to the vessel. b. After the pluripotent stem cells adhere to the culture medium, change the culture medium to the first culture medium, change the culture medium to the second culture medium on the 3rd-4th day, change the culture medium to the third culture medium on the 5th-6th day, change the culture medium to the fourth culture medium on the 7th-8th day, and change the culture medium to the fifth culture medium on the 10th-11th day. c. After obtaining monocytes through culture, culture the monocytes in the sixth medium for 2-3 days to obtain mature macrophages.

6. The method according to claim 5, characterized in that, The pluripotent stem cells include induced pluripotent stem cells.

7. The method according to claim 5, characterized in that, The pluripotent stem cells mentioned are gene-edited pluripotent stem cells.

8. The method according to claim 5, characterized in that, The culture container is coated with Vitronectin, Laminin 521, or iMatrix-511.

9. The method according to claim 5, characterized in that, The concentration of the pluripotent stem cells seeded was 0.5 × 10⁻⁶. 3 / cm 2 -5×10 3 / cm 2 .

10. The method according to claim 5, characterized in that, The concentration of the mononuclear cells seeded into the sixth culture medium was 0.5 × 10⁻⁶. 3 / cm 2 -5×10 3 / cm 2 .

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