Cell molecule activation method based on aqueous two-phase system

Abstract

The invention relates to the technical field of cytobiology and immunology, and discloses a cell molecule activation method based on an aqueous two-phase system, which comprises the following steps: preparing the aqueous two-phase system with the concentration of 2-15wt%; adding a suspension containing target cell molecules into the aqueous two-phase system, performing centrifugal separation, and concentrating the target cell molecules; and adding an exogenous signal molecule into the concentrated target cell molecule to activate the target cell molecule. The method has a wide application prospect in the fields of cell therapy, regenerative medicine, tumor immunotherapy and the like. By rapidly activating and enhancing cell functions, the effect of cell therapy can be improved, and a new method is provided for personalized medical treatment and immunotherapy.

Description

[0001] This invention relates to the fields of cell biology and immunology, and more specifically, to a method for cell and molecular activation based on an aqueous two-phase system. Background Technology

[0002] Stem cells, T cells, NK cells, macrophages, and B cells play crucial roles in immune responses and tissue repair. The functional activation and enhancement of these cells are central to many clinical applications, such as cancer immunotherapy, tissue engineering, and immune modulation. However, existing cell activation technologies are often time-consuming, inefficient, and may require complex culture procedures. Summary of the Invention

[0003] The purpose of this invention is to provide a cell and molecular activation method based on an aqueous two-phase system.

[0004] To achieve the above objectives, the present invention provides a cell and molecular activation method based on an aqueous two-phase system, comprising the following steps: Configure a two-phase aqueous system with a concentration of 2-15 wt%; The suspension containing the target cell molecules is added to the aqueous two-phase system, centrifuged, and the target cell molecules are concentrated. The target cell molecules are activated by adding exogenous signaling molecules to the concentrated target cell molecules.

[0005] Preferably, components A and B in the aqueous two-phase system are any one of dextran / polyethylene glycol (DEX / PEG), dextran / fish gelatin (DEX / fish gelatin), dextran / sucrose (DEX / FICOLL), and sucrose / polyethylene glycol (FICOLL / PEG).

[0006] Preferably, the mass fraction of component A is 6 wt% or 4 wt%, and the mass fraction of component B is 4 wt%, 6 wt%, 8 wt%, or 10 wt%.

[0007] Preferably, the mass fraction of component A is 6 wt%, and the mass fraction of component B is 6 wt%.

[0008] Preferably, in the aqueous two-phase system, component A: component B = 1:(1-100).

[0009] Preferably, in the aqueous two-phase system, component A: component B = 1:1.

[0010] Preferably, the target cell molecule is any one of stem cells, T cells, NK cells, macrophages, and B cells.

[0011] Preferably, the exogenous signal molecule is one or more of CD3, CD28, CD15, TLR1, TLR4, and TLR5.

[0012] Preferably, the centrifugation rate during the centrifugation separation is 200-500g.

[0013] Preferably, the activation conditions for the target cell molecule are 37°C and a CO2 concentration of 5%.

[0014] The aqueous two-phase system (ATPS) of this invention provides a relatively mild environment, reducing cell damage. Cellular molecules accumulate at the two-phase interface, increasing opportunities for intercellular contact. For immune cells, this dense contact can mimic in vivo immune contact, promoting intercellular signal transduction. Furthermore, the high concentration of cellular molecules in ATPS creates a molecularly crowded environment, which can enhance protein-protein and protein-ligand interactions, thereby accelerating signal transduction. Activating factors in cellular molecules (such as cytokines and growth factors) may accumulate in certain areas; this locally high concentration environment can accelerate and enhance cellular responses. In addition, ATPS affects cell membrane fluidity and receptor distribution, leading to receptor aggregation and thus enhancing signal transduction efficiency. ATPS also affects the metabolic state of cells, and under certain conditions, can promote cells to enter a more active metabolic state, facilitating rapid activation.

[0015] The beneficial effects of this invention are as follows: The aqueous two-phase system of this invention adds a suspension containing target cells to an aqueous two-phase system, utilizing phase separation characteristics to achieve concentration of target cell molecules through simple centrifugation or separation operations. Different aqueous two-phase combinations can selectively concentrate different types of cell molecules and components. The DEX / PEG system is suitable for concentrating cell components of specific sizes and charges, while the DEX / FISH gelatin system is suitable for enriching complex cell components.

[0016] This method induces stem cell proliferation and directed differentiation, making it suitable for regenerative medicine and tissue engineering; it can enhance the immune response of T cells, making it suitable for cancer immunotherapy; it can rapidly activate the cytotoxic activity of NK cells, applicable to tumor immunotherapy and viral infection treatment; it can enhance the phagocytic and anti-inflammatory activity of macrophages, contributing to tissue repair and inflammation control; and it can also promote the antibody production capacity of B cells, enhancing humoral immune responses. The aqueous two-phase system provided by this invention can achieve cellular molecule concentration and functional activation within minutes, significantly shortening experimental time.

[0017] This invention has broad application prospects in cell therapy, regenerative medicine, and tumor immunotherapy. By rapidly activating and enhancing cell function, it can improve the efficacy of cell therapy and provide new methods for personalized medicine and immunotherapy.

[0018] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0019] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the following detailed description to explain the invention, but do not constitute a limitation thereof. In the drawings: Figure 1 This illustrates, in one embodiment of the present invention, the distribution of Anti-CD3 antibody in a DEX / PEG aqueous two-phase system at different ratios and mass fractions; Figure 2 This illustrates, in one embodiment of the present invention, the distribution of Anti-CD28 antibody in a DEX / PEG aqueous two-phase system at different ratios and mass fractions; Figure 3 This invention illustrates the expression of IL-2 RNA in T cells at different time points and under different conditions in one embodiment of the invention; Figure 4 The expression of activating factors of T cells at different time points and under different conditions is shown in an embodiment of the present invention; Figure 5 The expression of macrophage activating factors at different time points and under different conditions is shown in an embodiment of the present invention; Figure 6 This illustration shows the distribution of Anti-CD3 antibody in a high-proportion DEX / PEG aqueous two-phase system according to one embodiment of the present invention. Figure 7 This illustration shows the distribution of Anti-CD28 antibody in a high-proportion DEX / PEG aqueous two-phase system according to one embodiment of the present invention. Detailed Implementation

[0020] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0021] Example 1 (using the DEX / PEG system as an example) This embodiment discloses a cell and molecular activation method based on an aqueous two-phase system, including the following steps: S1: Configure DEX / PEG aqueous two-phase systems with different mass fractions and ratios to investigate the partitioning of Anti-CD3 and Anti-CD28 antibodies in DEX / PEG aqueous two-phase systems with different mass fractions and ratios (e.g., ...). Figure 1 As shown and Figure 2(As shown). Different mass fractions were DEX:PEG = 6wt%:4wt%, 6wt%:6wt%, 6wt%:8wt%, and 6wt%:10wt%, and different ratios were DEX:PEG = 1:1, 1:2, 1:5, and 1:10. (From...) Figure 1 It can be seen that for Anti-CD3 antibody, the optimal mass fraction ratio of the DEX / PEG aqueous two-phase system is 6wt%:6wt%, and the optimal ratio is 1:1. For Anti-CD28 antibody, the concentration of Anti-CD28 antibody is relatively high when the mass fraction ratio of the DEX / PEG aqueous two-phase system is 4wt%:8wt%, with a ratio of 1:2 and 4wt%:6wt%, with a ratio of 1:5.

[0022] S2: Add the suspension of human peripheral blood T cells to the DEX / PEG aqueous two-phase system, and centrifuge (centrifugation rate 200g) to separate T cell-related molecules at a temperature of 37℃ and a CO2 concentration of 5%, achieving molecular enrichment of T cells within 5 minutes.

[0023] S3: Add co-stimulatory molecule yeast polysaccharide for activation.

[0024] S4: Detect the expression levels of T cell activating factors. These T cell activating factors include IL-2, CD69, CD25, IFN-γ, CD71, TNFα, and CD154.

[0025] Example 2 (Investigating the partitioning of Anti-CD3 antibody and Anti-CD28 antibody in a high-proportion DEX / PEG aqueous two-phase system) DEX / PEG aqueous two-phase systems with mass fractions of 6wt%:6wt% and ratios of 1:25, 1:50, and 1:100 were prepared to investigate the partitioning of Anti-CD3 and Anti-CD28 antibodies in high-proportion DEX / PEG aqueous two-phase systems. Figure 6 and Figure 7 As shown. By Figure 6 and Figure 7 It can be seen that when the ratio of DEX to PEG reaches 1:25 or higher, the Anti-CD3 antibody and Anti-CD28 will be further concentrated in the DEX / PEG aqueous two-phase system, and the degree of concentration is much higher than that of the low-ratio DEX / PEG aqueous two-phase system.

[0026] Comparative Example 1 The implementation steps in Comparative Example 1 are largely the same as those in Example 1, except that conventional cell culture plates (TCPS) are used for culture, and the expression levels of T cell activating factors are also detected. The expression levels of activating factors in Example 1 are compared with those in Comparative Example 1. Figure 3 and Figure 4 As shown.

[0027] Depend on Figure 3 and Figure 4 As can be seen, the expression level of T cell activating factors in the DEX / PEG aqueous two-phase system of this embodiment is much higher than that of the traditional culture plate at any time point (1h, 4h, 12h, 48h). This shows that the DEX / PEG aqueous two-phase system of this embodiment can rapidly concentrate the activating factors in T cells (in the figure, ...CD281, ...CD282 indicate that two experiments were performed).

[0028] Example 3 (using the DEX / FISH gelatin system as an example) S1: Configure a DEX / FISH gelatin two-phase aqueous system, wherein the mass fraction ratio of DEX / FISH gelatin is 4wt%:8wt%, and the ratio is 1:2.

[0029] S2: Macrophages were added to a DEX / FISH gelatin aqueous two-phase system. The active molecules of macrophages were separated by centrifugation (centrifugation rate 500g) at a temperature of 37℃ and a CO2 concentration of 5%, and the molecular enrichment of T cells was achieved within 10 minutes.

[0030] S3: Activation is achieved by adding co-stimulatory molecules CD3 and CD28.

[0031] S4: Detect the expression levels of active molecules in macrophages. Active molecules include IL-1β, NF-κB, and TNFα, etc.

[0032] Comparative Example 2 The implementation steps in Comparative Example 2 are largely the same as those in Example 3, except that conventional cell culture plates (TCPS) are used for culture, and the expression levels of macrophage activating factors are also detected. The expression levels of activating factors in Example 1 are compared with the expression levels of T cell activating factors in Comparative Example 1. Figure 5 As shown.

[0033] Comparative Example 3 The implementation steps in Comparative Example 3 were largely the same as those in Example 3, except that yeast polysaccharide (zymosan) was used for culturing, and the expression levels of macrophage activating factors were also detected. The expression levels of activating factors in Example 1 were compared with the expression levels of T cell activating factors in Comparative Example 1. Figure 5 As shown.

[0034] Depend on Figure 5As can be seen, the expression level of T cell activating factors in the DEX / FISH gelatin aqueous two-phase system of this embodiment is much higher than that of the traditional culture plate at 1h-4h. This shows that the DEX / FISH gelatin aqueous two-phase system of this embodiment can rapidly concentrate the active factors in macrophages.

[0035] The preferred embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the specific details of the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention. Furthermore, it should be noted that the various specific technical features described in the above embodiments can be combined in any suitable manner without contradiction. To avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0036] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A method for cell and molecular activation based on an aqueous two-phase system, characterized in that, Includes the following steps: Configure a two-phase aqueous system with a concentration of 2-15 wt%; The suspension containing the target cell molecules is added to the aqueous two-phase system, centrifuged, and the target cell molecules are concentrated. An exogenous signaling molecule is added to the concentrated target cell molecule to activate it.

2. The cell and molecular activation method based on an aqueous two-phase system according to claim 1, characterized in that, In the aqueous two-phase system, component A and component B are any one of dextran / polyethylene glycol, dextran / FISH gelatin, dextran / sucrose, and sucrose / polyethylene glycol.

3. The cell and molecular activation method based on an aqueous two-phase system according to claim 2, characterized in that, The mass fraction of component A is 6 wt% or 4 wt%, and the mass fraction of component B is 4 wt%, 6 wt%, 8 wt%, or 10 wt%.

4. The cell and molecular activation method based on an aqueous two-phase system according to claim 2, characterized in that, The mass fraction of component A is 6 wt%, and the mass fraction of component B is 6 wt%.

5. The cell and molecular activation method based on an aqueous two-phase system according to claim 3, characterized in that, In the aqueous two-phase system, component A: component B = 1: (1-100).

6. The cell and molecular activation method based on an aqueous two-phase system according to claim 5, characterized in that, In the aqueous two-phase system, component A: component B = 1:

1.

7. The cell and molecular activation method based on an aqueous two-phase system according to claim 1, characterized in that, The target cell molecule is any one of stem cells, T cells, NK cells, macrophages, and B cells.

8. The cell and molecular activation method based on an aqueous two-phase system according to claim 1, characterized in that, The exogenous signal molecules are one or more of CD3, CD28, CD15, TLR1, TLR4, and TLR5.

9. The cell and molecular activation method based on an aqueous two-phase system according to claim 1, characterized in that, The centrifugation rate during the centrifugation separation is 200-500g.

10. The cell and molecular activation method based on an aqueous two-phase system according to claim 1, characterized in that, The activation conditions for the target cell molecules are 37°C and a CO2 concentration of 5%.

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