Mesenchymal stem cell digestion solution, preparation method and application thereof

Abstract

The present application relates to a kind of mesenchymal stem cell digestive juice and its preparation method and application, belong to stem cell technical field.The mesenchymal stem cell of the present application includes trypsin, cell protective agent and phosphate buffer solution, in the digestive juice, the concentration of trypsin is 0.02-0.07wt%, the concentration of cell protective agent is 2.01005-7.05005wt%;The cell protective agent is the mixed solution of vitamin C, epidermal growth factor and serum albumin solution.The mesenchymal stem cell digestive juice of the present application, low concentration trypsin is combined with cell protective agent, can be fully dissociated outside mesenchymal stem cell while ensuring that cell is not over-digested by trypsin, and need not to add other reagent to terminate digestion, avoid other animal source serum to contaminate mesenchymal stem cell, reduce the culture cost of mesenchymal stem cell, simplify the culture step of mesenchymal stem cell.

Description

Technical Field

[0001] This invention relates to the field of stem cell technology, and in particular to a digestive solution for mesenchymal stem cells, its preparation method, and its application. Background Technology

[0002] Mesenchymal stem cells (MSCs) are a type of cell with self-renewal and multipotent differentiation potential. Also known as pluripotent stromal cells, they are a type of pluripotent stem cell belonging to the mesoderm, primarily found in connective tissues and organ stroma, including bone marrow, umbilical cord, adipose tissue, amnion, dental pulp, and placenta. Under suitable conditions, they can differentiate into various tissue cells such as fat, bone, and cartilage. MSCs are highly suitable for clinical research and application due to their convenient sourcing, lack of ethical controversy, large available cell numbers, strong proliferative capacity, significant immunomodulatory effects, and high levels of secreted cell growth factors. They are also easy to expand and passage, and do not present problems such as matching or rejection. MSCs are currently a research hotspot, which means that a large number of cells are needed for scientific experiments.

[0003] Due to the inherent characteristics of mesenchymal stem cell culture, the cultured cells need to be dissociated and prepared into a cell suspension before clinical research and application. Enzymatic digestion is one of the commonly used methods for cell dissociation. Trypsin (Parenzyme) is a serine proteolytic enzyme, often prepared as a 0.25% trypsin-EDTA solution in cell culture. This solution can be used as a cell digestion fluid to hydrolyze proteins between cells, thereby dissociating the cells. It can be used for tissues and monolayer adherent cells.

[0004] The cell-dispersing activity of trypsin solution is related to its concentration, temperature, and reaction time. The trypsin solution exhibits the strongest activity at pH 8.0 and 37°C. While concentration and temperature can be controlled relatively accurately, the reaction time largely depends on the experimenter's experience. For trypsin-sensitive cells such as mesenchymal stem cells, controlling the reaction time is even more difficult. Excessive reaction time leading to over-digestion can cause irreversible damage to precious stem cells, resulting in their loss.

[0005] Current trypsin digestion solutions require animal-derived serum or serum-containing culture media to terminate digestion because serum proteins bind to trypsin, competitively reducing its digestive effect on cells. However, the mainstream mesenchymal stem cell culture system has now shifted to animal-derived serum-free culture. Adding serum or serum-containing culture media to stem cells cultured in an animal-derived serum-free system will cause the stem cells to be affected by the animal-derived serum, thereby affecting stem cell growth and cell state, resulting in cell loss.

[0006] Trypsin is used in multiple steps of mesenchymal stem cell culture, from primary culture to passage culture. However, to date, there is still no digestive solution containing trypsin that can safely dissociate cells, stop digestion without the need for additional serum, and protect cells from waste, making it suitable for use with mesenchymal stem cells. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a digestive solution for mesenchymal stem cells that can fully dissociate mesenchymal stem cells while protecting them, and which does not require the addition of serum culture medium to terminate digestion, as well as its preparation method and application.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] In a first aspect, the present invention provides a digestive solution for mesenchymal stem cells, comprising trypsin, a cell protectant and phosphate buffer, wherein the concentration of trypsin in the digestive solution is 0.02-0.07 wt% and the concentration of the cell protectant is 2.01005-7.05005 wt%.

[0010] The cell protectant is a mixture of vitamin C, epidermal growth factor, and serum albumin solution; the weight ratio of vitamin C, epidermal growth factor, and serum albumin solution is vitamin C: epidermal growth factor: serum albumin solution = (0.01-0.05): (0.00001-0.00005): (2-7); the pH value of the digestive solution used for mesenchymal stem cells is 6.8-7.4.

[0011] This invention, by selecting a specific cell protectant and combining it with a low concentration of trypsin, produces a digestive solution for mesenchymal stem cells that has the advantages of gentle dissociation, protection of stem cells, and no need to add serum culture medium to terminate digestion.

[0012] This invention uses a combination of vitamin C, epidermal growth factor, and serum albumin as a cell protectant, which can prevent free radicals generated by trypsin digestion from damaging stem cells, maintain the original morphology of stem cells, maintain cell membrane function, maintain cell osmotic pressure, and terminate the effect of trypsin digestion in a timely manner.

[0013] In a preferred embodiment of the digestive solution for mesenchymal stem cells described in this invention, the digestive solution contains trypsin at a concentration of 0.04-0.06 wt% and a cell protectant at a concentration of 4.01001-6.03003 wt%; the weight ratio of vitamin C, epidermal growth factor, and serum albumin solution is vitamin C:epidermal growth factor:serum albumin solution = (0.01-0.03):(0.00001-0.00003):(4-6). Within this preferred ratio range, the digestive solution for mesenchymal stem cells of this invention is gentler, has better cell dissociation and protection effects.

[0014] In a preferred embodiment of the digestive solution for mesenchymal stem cells described in this invention, the digestive solution contains 0.05 wt% trypsin and 5.01001 wt% cell protectant; the weight ratio of vitamin C, epidermal growth factor, and serum albumin solution is 0.01:0.00001:5. Within this preferred ratio range, the digestive solution for mesenchymal stem cells of this invention exhibits the best effect in dissociating and protecting cells.

[0015] In a preferred embodiment of the digestive solution for mesenchymal stem cells described in this invention, the serum albumin solution contains human serum albumin; the concentration of serum albumin in the serum albumin solution is 20 wt%.

[0016] Serum albumin, an albumin extracted from animal plasma, plays a role in maintaining cell osmotic pressure and can exert a slight antagonistic effect on trypsin, thus terminating trypsin digestion to a certain extent. Therefore, the digestive solution for mesenchymal stem cells in this invention does not require the addition of serum-containing culture medium to terminate digestion. Furthermore, serum albumin can act as a carrier to bind with vitamin C and epidermal growth factor, enhancing their cell-protective effects during digestion. A 20wt% serum albumin solution is added to the digestive solution system, resulting in a final concentration of 0.4-1.4wt%, which is close to the concentration of mesenchymal stem cells in tissues, thus better maintaining cellular osmotic pressure balance.

[0017] In a preferred embodiment of the digestive fluid for mesenchymal stem cells described in this invention, the epidermal growth factor is recombinant human epidermal growth factor.

[0018] Epidermal growth factor (EGF) is a small polypeptide widely found in animal cells. Through genetic engineering, EGF from different sources can be heterologously expressed in hosts such as microorganisms, plants, and animal cells. Recombinant EGF has the same structure and active sites as natural EGF and plays a role in maintaining cell morphology and cell membrane function in digestive fluids. Combining it with vitamin C and serum albumin can further enhance its cell-protective effect. Furthermore, recombinant EGF is relatively inexpensive, which can further reduce the culture cost of mesenchymal stem cells.

[0019] Secondly, the present invention provides a method for preparing the digestive solution for mesenchymal stem cells, which includes the following steps: mixing a cell protectant with a phosphate buffer to obtain a mixture; mixing the obtained mixture with trypsin at 0-4°C to obtain a digestive solution for mesenchymal stem cells.

[0020] Thirdly, the present invention provides the application of the above-mentioned digestive solution for mesenchymal stem cells in the digestion of human umbilical cord mesenchymal stem cells.

[0021] Fourthly, the present invention provides a method for dissociating mesenchymal stem cells, comprising the following steps: removing the culture medium from the mesenchymal stem cells, adding the above-mentioned digestive solution for mesenchymal stem cells, digesting for 2-30 minutes, and obtaining a cell suspension of mesenchymal stem cells.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] (1) The digestive solution for mesenchymal stem cells of the present invention uses a low concentration of trypsin combined with a cell protectant, which can fully dissociate mesenchymal stem cells while ensuring that the cells are not over-digested by trypsin, and does not require the addition of other reagents to terminate digestion, thus avoiding contamination of mesenchymal stem cells by other animal-derived serum, reducing the culture cost of mesenchymal stem cells, and simplifying the culture steps of mesenchymal stem cells.

[0024] (2) This invention uses vitamin C, epidermal growth factor, and serum albumin as cell protectants to maintain the cell activity, cell membrane function, and cell osmotic pressure of mesenchymal stem cells, thus maintaining a dynamic balance. Furthermore, serum albumin has a slight antagonistic effect with trypsin, inhibiting its enzymatic activity to a certain extent, thereby terminating digestion without the need for serum culture medium. The three ingredients of the cell protectant work synergistically to protect the cells, preventing prolonged enzymatic digestion from damaging the membrane proteins of mesenchymal stem cells and leading to their loss. Attached Figure Description

[0025] Figure 1Microscopic images of mesenchymal stem cells digested for 2 min and 30 min with the digestive solutions of Example 1 and Comparative Example 1 of the present invention, respectively; and microscopic images of mesenchymal stem cells digested for 30 min with the digestive solutions of Examples 2-5 and Comparative Examples 2-3. Detailed Implementation

[0026] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0027] In the following examples and comparative examples, recombinant human epidermal growth factor was provided by abcam, catalog number ab259398-10μg;

[0028] 20wt% human serum albumin solution was supplied by Behring, catalog number S20140078;

[0029] The fluorescent primary antibodies—mouse anti-human CD73-APC monoclonal antibody, mouse anti-human CD90-PerCP-Cyanine 5.5 monoclonal antibody, mouse anti-human CD105-PE-Cyanine 7 monoclonal antibody, mouse anti-human CD19-PE monoclonal antibody, mouse anti-human CD45-PE monoclonal antibody, mouse anti-human CD34-PE monoclonal antibody, mouse anti-human CD11b-PE monoclonal antibody, and mouse anti-human HLA-DR-PE monoclonal antibody—were provided by Biolegend.

[0030] All other reagents and consumables can be obtained commercially.

[0031] Example 1

[0032] This invention provides an embodiment of a digestive solution for mesenchymal stem cells and its preparation method. The digestive solution for mesenchymal stem cells in this embodiment comprises the following components and their amounts, as shown in Table 1: 0.05g trypsin, 0.01g vitamin C, 0.00001g epidermal growth factor, 5ml 20wt% serum albumin solution, and 95ml phosphate buffer. The epidermal growth factor is recombinant human epidermal growth factor, the serum albumin in the 20wt% serum albumin solution is human serum albumin, and the phosphate buffer has a concentration of 0.01M and a pH of 6.8-7.2. Other embodiments and comparative examples do not have specific descriptions, and the types of components used are the same as in this embodiment.

[0033] The method for preparing the digestive solution for mesenchymal stem cells described in this embodiment is as follows: phosphate buffer and serum albumin solution are mixed, vitamin C and epidermal growth factor are added and mixed to obtain a mixture; the resulting mixture is stirred with trypsin at 4°C for 1 hour, and the pH is adjusted to 6.8-7.2 with 0.1M hydrochloric acid or 0.1M sodium hydroxide to obtain the digestive solution for mesenchymal stem cells.

[0034] Table 1. Components and dosages of digestive solutions used for mesenchymal stem cells in Examples 1-5 and Comparative Example 3.

[0035]

[0036] Example 2

[0037] This invention provides an embodiment of a digestive solution for mesenchymal stem cells and its preparation method. The components and amounts of the digestive solution for mesenchymal stem cells described in this embodiment are shown in Table 1. The preparation method is the same as that of the digestive solution in Example 1.

[0038] Example 3

[0039] This invention provides an embodiment of a digestive solution for mesenchymal stem cells and its preparation method. The components and amounts of the digestive solution for mesenchymal stem cells described in this embodiment are shown in Table 1. The preparation method is the same as that of the digestive solution in Example 1.

[0040] Example 4

[0041] This invention provides an embodiment of a digestive solution for mesenchymal stem cells and its preparation method. The components and amounts of the digestive solution for mesenchymal stem cells described in this embodiment are shown in Table 1. The preparation method is the same as that of the digestive solution in Example 1.

[0042] Example 5

[0043] This invention provides an embodiment of a digestive solution for mesenchymal stem cells and its preparation method. The components and amounts of the digestive solution for mesenchymal stem cells described in this embodiment are shown in Table 1. The preparation method is the same as that of the digestive solution in Example 1.

[0044] Comparative Example 1

[0045] This invention provides a comparative example of a digestive solution for mesenchymal stem cells and its preparation method. The digestive solution for mesenchymal stem cells described in this comparative example is composed of the following components in parts by weight: 0.25g trypsin and 100ml ethylenediaminetetraacetic acid solution; wherein the ethylenediaminetetraacetic acid solution is 0.3mM and has a pH of 7.4.

[0046] The method for preparing the digestive solution for mesenchymal stem cells described in this comparative example is as follows: trypsin and ethylenediaminetetraacetic acid solution are mixed at 4°C to obtain the digestive solution for mesenchymal stem cells.

[0047] Comparative Example 2

[0048] This invention provides a comparative example of a digestive solution for mesenchymal stem cells and its preparation method. The composition of the digestive solution for mesenchymal stem cells described in this comparative example is similar to that in Example 1, except that serum albumin solution is not added, while the remaining components, their amounts, and parameters remain unchanged.

[0049] The preparation method of the digestive fluid for mesenchymal stem cells described in this comparative example is the same as that in Example 1.

[0050] Comparative Example 3

[0051] This invention provides a comparative example of a digestive solution for mesenchymal stem cells and its preparation method. The components and amounts of the digestive solution for mesenchymal stem cells described in this comparative example are shown in Table 1. The preparation method is the same as in Example 1. Effect Example: The influence of the digestive solution on mesenchymal stem cells.

[0052] Experimental subjects: human umbilical cord mesenchymal stem cells (MSCs).

[0053] Experimental samples: digestion solutions from Examples 1-5 and Comparative Examples 1-3. The digestion solutions mentioned above need to be filtered and sterilized using a 0.22μm filter membrane before use.

[0054] P3 generation MSCs were seeded into T75 culture flasks containing 15 ml of culture medium and cultured at 37°C in a 5% CO2 incubator until the cell density reached 90%. The culture medium was then removed, and 3 ml of digestion solution was added to each flask to obtain a cell suspension of human umbilical cord mesenchymal stem cells. The different digestion solutions and digestion times are grouped as shown in Table 2.

[0055] Table 2. Treatment groups based on different digestive solutions and digestion times

[0056]

[0057] The cell suspension was transferred to centrifuge tubes, centrifuged at 500g for 5 minutes, the supernatant was discarded, and the cells were resuspended in 10ml of physiological saline. Cell morphology was observed under a light microscope, and cell viability and cell surface markers were measured. The results of the light microscope observations are shown below. Figure 1 .

[0058] like Figure 1As shown, the morphology of MSCs digested for 30 minutes with the digestive solutions of Examples 1-5 was not significantly different from that of MSCs digested for 2 minutes with the digestive solution of Example 1. The cells were in good condition, round in shape, with clear cell membrane edges and no cell clumping. The morphology of MSCs digested for 30 minutes with the digestive solution of Comparative Example 1 differed significantly from that of MSCs digested for 2 minutes with the digestive solution of Comparative Example 1. The MSCs digested for 30 minutes were in poor condition, with severe intercellular adhesion and cell membrane damage, resulting in most cells becoming polygonal. The morphology of MSCs digested for 30 minutes with the digestive solutions of Comparative Examples 2-3 differed significantly from that of MSCs digested for 2 minutes with the digestive solution of Example 1. The MSCs digested for 30 minutes were in poor condition, with severe intercellular adhesion and cell membrane damage, resulting in most cells becoming polygonal. This indicates that the digestive solution for mesenchymal stem cells of the present invention can be used for mesenchymal stem cells requiring long-term digestion without the need for additional serum-containing culture medium to terminate digestion, thus saving experimental costs.

[0059] Cell viability was determined using the trypan blue staining method. The centrifuged cell suspension was mixed with 0.4% trypan blue solution at a volume ratio of 9:1. After staining for 3 minutes, the mixture was transferred to a cell counter to count live and dead cells, and cell viability was calculated. The results are shown in Table 3. Cell viability (%) = (Total number of cells - Number of blue cells) / Total number of cells × 100%.

[0060] Table 3 Effects of different treatments on the viability of mesenchymal stem cells

[0061]

[0062] As shown in Table 3, the digestion solution treatment method for MSCs in the control group had no effect on cell viability. Compared with Comparative Examples 1-3, MSCs in the experimental groups (Examples 1-5) maintained good cell viability after 30 minutes of treatment with the digestion solution in Examples 1-5, and their cell viability was close to that of the control group. This indicates that the digestion solution of the present invention has a good digestion effect, and prolonged digestion treatment does not cause a decrease in cell viability. Compared with the digestion solution in Comparative Example 2, MSCs treated with the digestion solution in Example 1 maintained good cell viability, while the cell viability of MSCs treated with the digestion solution in Comparative Example 2 was only 55.37%. This shows that adding serum albumin can effectively dissociate MSCs while maintaining their cell viability, avoiding cell viability damage due to excessive dissociation. Compared with the digestion solution of Comparative Example 3, MSCs treated with the digestion solution of Example 1 maintained good cell viability, while the cell viability of MSCs treated with the digestion solution of Comparative Example 2 was only 68.7%. This shows that when the weight ratio of vitamin C, epidermal growth factor and serum albumin solution in the digestion solution of the present invention is (0.01-0.05):(0.00001-0.00005):(2-7), it can effectively and gently dissociate MSCs while protecting the cells and avoiding loss of MSC viability.

[0063] Cell surface markers were measured using flow cytometry. Centrifuged cell suspensions were mixed with antibodies and then analyzed by flow cytometry. The results are shown in Table 4. The antibodies used included CD73, CD90, CD105, CD19, CD45, CD34, CD11b, and HLA-DR.

[0064] Table 4. Effects of different treatments on surface markers of mesenchymal stem cells.

[0065]

[0066] As shown in Table 4, in the control group, MSCs treated with the digestion solutions of Example 1 and Comparative Example 1 showed normal expression of cell surface markers, indicating that shorter digestion times do not lead to a decline in MSC function. In the experimental groups (Examples 1-5), compared to Comparative Example 1, MSCs treated with the digestion solution of Example 1 showed normal expression of cell surface markers and normal cell function, while MSCs treated with the digestion solution of Comparative Example 1 showed a significant decrease in cell surface marker expression. This demonstrates that the digestion solution of the present invention does not cause a decline in MSC function during prolonged digestion, and can gently and effectively dissociate MSCs while protecting cell function. Compared with Comparative Examples 2-3, in the experimental group, MSCs treated with the digestion solution of Example 1 showed normal expression of cell surface markers and normal cell function. The expression of cell surface markers in MSCs treated with the digestion solution of Comparative Examples 2 or 3 was significantly reduced, and the expression in Comparative Example 2 was lower than that in Comparative Example 3. This indicates that when the weight ratio of serum albumin and vitamin C, epidermal growth factor and serum albumin solution in the digestion solution is (0.01-0.05):(0.00001-0.00005):(2-7), it can effectively protect MSCs from over-digestion and avoid MSC loss during digestion.

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A digestive solution for mesenchymal stem cells, characterized in that, The digestive solution consists of trypsin, a cell protectant, and phosphate buffer. The concentration of trypsin in the digestive solution is 0.02-0.07 wt%, and the concentration of the cell protectant is 2.01001-7.05005 wt%. The cell protectant is a mixture of vitamin C, epidermal growth factor, and serum albumin solution; the weight ratio of vitamin C, epidermal growth factor, and serum albumin solution is vitamin C: epidermal growth factor: serum albumin solution = (0.01-0.05):(0.00001-0.00005):(2-7); the pH value of the digestive solution used for mesenchymal stem cells is 6.8-7.

4.

2. The digestive solution for mesenchymal stem cells as described in claim 1, characterized in that, The digestive fluid contains 0.04-0.06 wt% trypsin and 4.01001-6.03003 wt% cell protectant. The weight ratio of vitamin C, epidermal growth factor and serum albumin solution is (0.01-0.03): (0.00001-0.00003): (4-6).

3. The digestive solution for mesenchymal stem cells as described in claim 2, characterized in that, The digestive fluid contains 0.05 wt% trypsin and 5.01001 wt% cell protectant. The weight ratio of vitamin C, epidermal growth factor and serum albumin solution is 0.01:0.00001:

5.

4. The digestive solution for mesenchymal stem cells as described in claim 1, characterized in that, The serum albumin in the serum albumin solution is human serum albumin; the concentration of serum albumin in the serum albumin solution is 20 wt%.

5. The digestive solution for mesenchymal stem cells as described in claim 1, characterized in that, The epidermal growth factor is recombinant human epidermal growth factor.

6. The method for preparing digestive fluid for mesenchymal stem cells according to any one of claims 1 to 5, characterized in that, Includes the following steps: The cell protectant was mixed with phosphate buffer to obtain a mixture; the resulting mixture was then mixed with trypsin at 0-4°C to obtain a digestion solution for mesenchymal stem cells.

7. The use of the digestive solution for mesenchymal stem cells as described in any one of claims 1 to 5 in the digestion of human umbilical cord mesenchymal stem cells.

8. A method for dissociating mesenchymal stem cells, characterized in that, Includes the following steps: Remove the culture medium from the mesenchymal stem cells, add the digestive solution for mesenchymal stem cells as described in any one of claims 1 to 5, and digest for 2-30 minutes to obtain a cell suspension of mesenchymal stem cells.

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