Culture dish for umbilical cord blood NK cells and use method of culture dish

Abstract

The invention belongs to the technical field of cell culture, and particularly relates to an umbilical cord blood NK cell culture dish and a use method thereof. The culture dish is reasonable in design, the bone marrow microenvironment is simulated through the three-dimensional micro-scaffold, and the adhesion and proliferation efficiency of cord blood NK precursor cells is remarkably improved by combining biocompatible nanofibers (PLGA) and extracellular matrix proteins (fibronectin and heparin sulfate), so that gt; the amplification multiple is 1 * 10 < 4 >. The dynamic microfluidic gradient channel continuously provides cell factors (such as IL-15 and IL-12) and nutrition, maintains stable glucose concentration (gt, 3mM) and low lactic acid level (lt, 15mM), and prevents metabolic waste from inhibiting amplification. According to the invention, an anti-CD16 antibody and an NKG2D ligand (ULBP2) are fixed on the surface, ADCC effect and tumor targeted killing ability of NK cells are enhanced through a costimulatory signal (CD137L), and cytotoxicity (K562 killing rate) gt is enhanced; 80%.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell culture, and particularly relates to a culture dish for umbilical cord blood NK cells and a method for using the same. Background Art

[0002] Natural killer cells (NK cells) are an important part of the human immune system. They are effector cells of the innate immune system, originating from bone marrow lymphoid stem cells, differentiating and maturing in the bone marrow and thymus, and are distributed in blood, lymph nodes, bone marrow, etc. Different from T and B cells, NK cells can non-specifically recognize and kill tumor cells and virus-infected cells without antigen sensitization and MHC restriction. Due to this important function, the research and application of NK cells in the field of tumor immunotherapy have been gradually taken seriously.

[0003] In clinical applications, NK cells have relatively high requirements for quantity and purity, especially purity, and should not contain excessive lymphocytes such as T cells and B cells. Since the lymphocyte ratio in peripheral blood varies greatly among individuals, the purity of NK cells cultured from PBMC extracted from peripheral blood using the same culture method is uneven, while the naive T cells contained in umbilical cord blood are not easily induced to cause graft-versus-host disease (GVHD), and at the same time, the lymphocyte ratio in umbilical cord blood is relatively stable, and the quality level of NK cells cultured by the same culture method is stable. Therefore, umbilical cord blood is an ideal source for preparing NK cells.

[0004] There is a lack of culture dishes suitable for culturing umbilical cord blood NK cells in the prior art. Summary of the Invention

[0005] The purpose of the present invention is to overcome the above problems existing in the traditional technology, and provide a culture dish for umbilical cord blood NK cells and a method for using the same.

[0006] To achieve the above technical objectives and reach the above technical effects, the present invention is realized through the following technical solutions: The present invention provides a culture dish for umbilical cord blood NK cells, including a culture dish body, a culture dish cover, a support ring, a hollowed-out support plate, a three-dimensional micro scaffold, a circular microfluidic network, an injection tube, an antibody co-stimulation array, and a detachable magnetic bead coupling layer. A support ring is installed at the bottom edge of the culture dish body, a hollowed-out support plate is installed above the support ring, a three-dimensional micro scaffold is installed above the hollowed-out support plate, a circular microfluidic network is sleeved and installed outside the three-dimensional micro scaffold, an injection tube passing through the culture dish body is connected to the side end of the circular microfluidic network, an antibody co-stimulation array is jointly installed above the three-dimensional micro scaffold and the circular microfluidic network, and a detachable magnetic bead coupling layer is installed inside the culture dish cover.

[0007] Furthermore, in the above-mentioned culture dish for umbilical cord blood NK cells, the inner area of ​​the support ring serves as a substrate installation area, and the substrate pre-installed in the substrate installation area adopts medical-grade polycarbonate or gas-permeable polymer, and the bottom of the substrate is integrated with a porous breathable membrane that ensures efficient oxygen / carbon dioxide exchange and supports high-density culture of umbilical cord blood NK cells.

[0008] Furthermore, in the above-mentioned culture dish for umbilical cord blood NK cells, the culture dish body, the culture dish cover, the support ring, and the hollow support plate are made of the same material, which is glass.

[0009] Furthermore, in the above-mentioned culture dish of umbilical cord blood NK cells, the three-dimensional microscaffold is a biocompatible nanofiber scaffold made of polylactic acid-glycolic acid copolymer, which can simulate the bone marrow microenvironment.

[0010] Furthermore, in the above-mentioned umbilical cord blood NK cell culture dish, the fiber diameter in the three-dimensional microscaffold is controlled at 200-500 nm, which can promote cell anchoring and migration; the surface of the three-dimensional microscaffold is coated with recombinant fibronectin and heparin sulfate for enhancing extracellular matrix adhesion signals.

[0011] Furthermore, in the above-mentioned culture dish for umbilical cord blood NK cells, the annular microfluidic network is connected to a syringe via a peristaltic pump to achieve continuous perfusion of the culture medium, and the syringe is equipped with a one-way valve.

[0012] Furthermore, in the above-mentioned umbilical cord blood NK cell culture dish, the culture medium comprises a basal culture medium and a cytokine sustained-release gel, the basal culture medium is X-VIVO 15 + 5% human AB serum; the cytokine sustained-release gel comprises IL-15, IL-12 and IL-18; the annular microfluidic network forms a cytokine concentration gradient from the outside to the inside to simulate the in vivo activation signal, and the cytokine concentration gradient decreases from 10 ng / mL to 1 ng / mL.

[0013] Furthermore, in the above-mentioned umbilical cord blood NK cell culture dish, the antibody co-stimulatory array is first fixed on the surface of the nanofiber with anti-CD16 single-chain antibody and NKG2D ligand ULBP2, and then connected through a flexible PEG spacer arm; the density of the antibody co-stimulatory array is 100-200 molecules / μm 2 , optimizing the signal activation threshold; the antibody co-stimulatory array can co-localize CD137L nanoclusters and enhance the persistent memory phenotype.

[0014] Furthermore, in the above-mentioned culture dish for umbilical cord blood NK cells, a magnetic grid is arranged on the top of the detachable magnetic bead coupling layer, and anti-CD56 / anti-CD3 magnetic beads are pre-loaded; Initial stage: Magnetic beads capture NK precursors in cord blood mononuclear cells, and the NK precursors are CD56+CD3-.

[0015] After amplification: Turn off the magnetic field to automatically release and purify NK cells.

[0016] The present invention also provides a method for using a culture dish for cord blood NK cells, comprising the following steps: 1) Inoculation: Inject 1×10 6 / mL of cord blood mononuclear cell suspension into the culture dish, and the magnetic bead layer captures NK precursors; 2) Amplification stage: Continuously perfuse the medium containing IL-15 / IL-12, and the microfluidic system maintains glucose > 3 mM and lactate < 15 mM; 3) Functional activation: Add K562-mbIL21 feeder cells on the 7th day, and the addition ratio is 1:5, and co-culture for 72 hours; 4) Harvesting: Lower the temperature to trigger cell detachment, and the magnetic bead layer is used for secondary purification to obtain >95% CD56+CD16+ NK cells.

[0017] The beneficial effects of the present invention are as follows: 1. High amplification ability: By simulating the bone marrow microenvironment with a three-dimensional micro-scaffold, combining biocompatible nanofibers (PLGA) and extracellular matrix proteins (fibronectin, heparin sulfate), the adhesion and proliferation efficiency of cord blood NK precursor cells are significantly improved, supporting an amplification multiple of >1×10 4 times within 21 days.

[0018] 2. Dynamic microfluidic gradient channels: Continuously provide cytokines (such as IL-15, IL-12) and nutrients, maintain a stable glucose concentration (>3 mM) and a low lactate level (<15 mM), and avoid the inhibition of amplification by metabolic wastes.

[0019] 3. Enhanced functional activity: Anti-CD16 antibody and NKG2D ligand (ULBP2) are surface-fixed, and the ADCC effect and tumor-targeted killing ability of NK cells are enhanced through co-stimulatory signals (CD137L), and the cytotoxicity (K562 killing rate) > 80%.

[0020] An integrated sensor monitors pH, lactate and cell impedance in real time, dynamically optimizes culture parameters, and reduces the expression of exhaustion markers (such as TIM-3) to <10%.

[0021] 4. Standardization and clinical application adaptability: The detachable magnetic bead layer enables efficient sorting of NK precursors in cord blood mononuclear cells (CD56+CD3- purity > 95%), while avoiding T cell contamination, meeting the low immunogenicity requirements for allogeneic treatment.

[0022] Of course, it is not necessary for any product implementing the present invention to achieve all of the above advantages simultaneously. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for describing the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can be obtained based on these drawings.

[0024] Figure 1 is a schematic structural diagram of the whole of the present invention; Figure 2 is a schematic structural diagram of the culture dish body of the present invention; Figure 3 is a schematic structural diagram of the hollow support plate and the three-dimensional micro scaffold of the present invention; Figure 4 is a front view schematic diagram of the annular microchannel network and the injection tube of the present invention; Figure 5 is a top view schematic diagram of the annular microchannel network and the injection tube of the present invention; Figure 6 is a schematic structural diagram of the culture dish cover and the detachable magnetic bead coupling layer of the present invention; In the accompanying drawings, the reference numerals of the various components are as follows: 1 - culture dish body, 2 - culture dish cover, 3 - support ring, 4 - hollow support plate, 5 - three-dimensional micro scaffold, 6 - annular microchannel network, 7 - injection tube, 8 - antibody co-stimulation array, 9 - detachable magnetic bead coupling layer. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0025] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, rather than all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art without creative efforts based on the embodiments of the present invention belong to the scope of protection of the present invention.

[0026] Such as Figures 1 - 6As shown in the figure, this embodiment provides a culture dish for umbilical cord blood NK cells, which includes a culture dish body 1, a culture dish cover 2, a support ring 3, a hollowed-out support plate 4, a three-dimensional micro scaffold 5, a circular microchannel network 6, an injection tube 7, an antibody co-stimulation array 8, and a detachable magnetic bead coupling layer 9. A support ring 3 is installed at the bottom edge of the culture dish body 1, a hollowed-out support plate 4 is installed on the upper side of the support ring 3, and a three-dimensional micro scaffold 5 is installed on the upper side of the hollowed-out support plate 4. A circular microchannel network 6 is sleeved and installed on the periphery of the three-dimensional micro scaffold 5. An injection tube 7 passing through the culture dish body 1 is connected to the side end of the circular microchannel network 6. An antibody co-stimulation array 8 is jointly installed on the upper sides of the three-dimensional micro scaffold 5 and the circular microchannel network 6, and a detachable magnetic bead coupling layer 9 is installed on the inner side of the culture dish cover 2.

[0027] In this embodiment, the inner area of the support ring 3 serves as a substrate installation area. The pre-installed substrate in the substrate installation area is made of medical-grade polycarbonate or a gas-permeable high molecular polymer. A porous breathable membrane that ensures efficient exchange of oxygen / carbon dioxide and supports high-density culture of umbilical cord blood NK cells is integrated at the bottom of the substrate.

[0028] In this embodiment, the culture dish body 1, the culture dish cover 2, the support ring 3, and the hollowed-out support plate 4 are made of the same material, which is glass.

[0029] In this embodiment, the three-dimensional micro scaffold 5 is a biocompatible nanofiber scaffold made of poly(lactic-co-glycolic acid) and can simulate the bone marrow microenvironment.

[0030] In this embodiment, the fiber diameter in the three-dimensional micro scaffold 5 is controlled within 200 - 500 nm, which can promote cell anchoring and migration; the surface of the three-dimensional micro scaffold 5 is coated with recombinant fibronectin and heparin sulfate for enhancing extracellular matrix adhesion signals.

[0031] In this embodiment, the circular microchannel network 6 realizes continuous perfusion of the culture medium by connecting the injection tube 7 through a peristaltic pump, and a one-way valve is built into the injection tube 7.

[0032] In this embodiment, the culture medium includes a basal medium and a cytokine sustained-release gel. The basal medium is X-VIVO15 + 5% human AB serum; the cytokine sustained-release gel contains IL-15, IL-12, and IL-18; the circular microchannel network 6 forms a cytokine concentration gradient for simulating in vivo activation signals from the outside to the inside, and the cytokine concentration gradient decreases from 10 ng / mL to 1 ng / mL.

[0033] In this embodiment, the antibody co-stimulation array 8 is formed by first immobilizing anti-CD16 single-chain antibody and NKG2D ligand ULBP2 on the surface of the nanofibers and then connecting them through a flexible PEG spacer arm; the density of the antibody co-stimulation array 8 is 100 - 200 molecules / μm2 Optimize the signal activation threshold; the antibody co-stimulation array 8 can co-localize CD137L nanoclusters and enhance the persistent memory phenotype.

[0034] In this embodiment, a magnetic grid is provided on the top of the detachable magnetic bead coupling layer 9, and anti-CD56 / anti-CD3 magnetic beads are pre-loaded; Initial stage: The magnetic beads capture NK precursors in umbilical cord blood mononuclear cells, and the NK precursors are CD56+CD3-.

[0035] After amplification: Turn off the magnetic field to automatically release and purify NK cells.

[0036] This embodiment also provides a method for using a culture dish for umbilical cord blood NK cells, including the following steps: 1) Inoculation: Inject 1×10 6 / mL of umbilical cord blood mononuclear cell suspension into the culture dish, and the magnetic bead layer captures NK precursors; 2) Amplification stage: Continuously perfuse the medium containing IL-15 / IL-12, and the microfluidic system maintains glucose > 3 mM and lactate < 15 mM; 3) Functional activation: Add K562-mbIL21 feeder cells on the 7th day, and the addition ratio is 1:5, and co-culture for 72 hours; 4) Harvesting: Lower the temperature to trigger cell detachment, and the magnetic bead layer is purified for the second time to obtain > 95% CD56+CD16+ NK cells.

[0037] A specific application of this embodiment is: Simulate the bone marrow microenvironment through a three-dimensional micro-scaffold, combine biocompatible nanofibers (PLGA) and extracellular matrix proteins (fibronectin, heparin sulfate), and significantly improve the adhesion and proliferation efficiency of umbilical cord blood NK precursor cells, supporting an amplification multiple of > 1×104 times within 21 days.

[0038] The dynamic microfluidic gradient channel continuously provides cytokines (such as IL-15, IL-12) and nutrients, maintains a stable glucose concentration (> 3 mM) and a low lactate level (< 15 mM), and avoids the inhibition of amplification by metabolic wastes.

[0039] Fix anti-CD16 antibody and NKG2D ligand (ULBP2) on the surface, enhance the ADCC effect and tumor-targeted killing ability of NK cells through co-stimulatory signals (CD137L), and the cytotoxicity (K562 killing rate) > 80%.

[0040] The integrated sensor monitors pH, lactate and cell impedance in real time, dynamically optimizes the culture parameters, and reduces the expression of exhaustion markers (such as TIM-3) to < 10%.

[0041] The detachable magnetic bead layer enables efficient sorting of NK precursors in umbilical cord blood mononuclear cells (CD56+CD3- purity > 95%), while avoiding T cell contamination, meeting the low immunogenicity requirements for allogeneic therapy.

[0042] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to only the specific embodiments. Obviously, many modifications and variations can be made according to the content of this specification. These embodiments are selected and specifically described in this specification in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can well understand and utilize the present invention. The present invention is only limited by the claims and their full scope and equivalents.

Claims

1. A culture dish for umbilical cord blood NK cells, characterized in that: It includes a culture dish body, a culture dish cover, a support ring, a hollow support plate, a three-dimensional micro-stent, an annular microfluidic network, an injection tube, an antibody co-stimulation array and a detachable magnetic bead coupling layer. A support ring is installed at the bottom edge of the culture dish body, a hollow support plate is installed on the upper side of the support ring, a three-dimensional micro-stent is installed on the upper side of the hollow support plate, an annular microfluidic network is sleeved and installed on the periphery of the three-dimensional micro-stent, a side end of the annular microfluidic network is connected to an injection tube running through the culture dish body, an antibody co-stimulation array is installed on the upper side of the three-dimensional micro-stent and the annular microfluidic network, and a detachable magnetic bead coupling layer is installed on the inner side of the culture dish cover.

2. The culture dish of umbilical cord blood NK cells according to claim 1, characterized in that: The inner area of ​​the support ring serves as a substrate installation area. The pre-installed substrate in the substrate installation area is made of medical-grade polycarbonate or gas-permeable polymer. The bottom of the substrate is integrated with a porous breathable membrane that ensures efficient oxygen / carbon dioxide exchange and supports high-density culture of umbilical cord blood NK cells.

3. The culture dish of umbilical cord blood NK cells according to claim 1, characterized in that: The culture dish body, the culture dish cover, the support ring and the hollow support plate are made of the same material, which is glass.

4. The culture dish of umbilical cord blood NK cells according to claim 1, characterized in that: The three-dimensional microscaffold is a biocompatible nanofiber scaffold made of poly(lactic acid-glycolic acid) copolymer, which can simulate the bone marrow microenvironment.

5. The culture dish for umbilical cord blood NK cells according to claim 4, characterized in that: The fiber diameter in the three-dimensional microscaffold is controlled at 200 to 500 nm, which can promote cell anchoring and migration; the surface of the three-dimensional microscaffold is coated with recombinant fibronectin and heparin sulfate for enhancing extracellular matrix adhesion signals.

6. The culture dish for umbilical cord blood NK cells according to claim 1, characterized in that: The annular microfluidic network is connected to an injection tube via a peristaltic pump to achieve continuous perfusion of culture medium, and the injection tube is equipped with a one-way valve.

7. The culture dish of umbilical cord blood NK cells according to claim 6, characterized in that: The culture medium comprises a basal culture medium and a cytokine sustained-release gel, wherein the basal culture medium is X-VIVO 15 + 5% human AB serum; the cytokine sustained-release gel comprises IL-15, IL-12 and IL-18; the annular microfluidic network forms a cytokine concentration gradient from the outside to the inside for simulating in vivo activation signals, and the cytokine concentration gradient decreases from 10 ng / mL to 1 ng / mL.

8. The culture dish for umbilical cord blood NK cells according to claim 1, characterized in that: The antibody co-stimulatory array is formed by first fixing the anti-CD16 single-chain antibody and the NKG2D ligand ULBP2 on the surface of the nanofiber, and then connecting them through a flexible PEG spacer arm; the density of the antibody co-stimulatory array is 100 to 200 molecules / μm 2 , optimizing the signal activation threshold; the antibody co-stimulatory array can co-localize CD137L nanoclusters and enhance the persistent memory phenotype.

9. The culture dish for umbilical cord blood NK cells according to claim 1, characterized in that: A magnetic grid is arranged on the top of the detachable magnetic bead coupling layer, and is pre-loaded with anti-CD56 / anti-CD3 magnetic beads; Initial stage: magnetic beads capture NK precursors in cord blood mononuclear cells, which are CD56+CD3-; after amplification: turn off the magnetic field to automatically release purified NK cells.

10. The method for using the culture dish of umbilical cord blood NK cells according to any one of claims 1 to 9, characterized in that: The steps include: 1) Inoculation: 1×10 6 / mL of cord blood mononuclear cell suspension was injected into the culture dish, and the magnetic bead layer captured NK precursors; 2) Expansion phase: Continuous perfusion of culture medium containing IL-15 / IL-12, and microfluidic system to maintain glucose>3 mM and lactate<15 mM; 3) Functional activation: K562-mbIL21 trophoblasts were added on day 7 at a ratio of 1:5 and cultured for 72 hours; 4) Harvest: Cooling triggers cell shedding, followed by secondary purification on the magnetic bead layer to obtain >95% CD56+CD16+ NK cells.

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