CN106381284B - Method for preparing stem cell active factor - Google Patents

Abstract

The invention relates to a method for preparing stem cell active factors. Specifically, one aspect of the present invention relates to a method for preparing a stem cell active factor, which comprises the steps of: (1) obtaining umbilical cord source mesenchymal stem cells; (2) expanding cells; (3) cell identification and detection; (4) preparing stem cell active factors; (5) optionally freeze drying. Further, the invention also relates to an exemplary lyophilized powder of the stem cell active factor prepared by the method. The method has the advantages as described in the specification of the invention.

Description

Method for preparing stem cell active factor

Technical Field

The invention belongs to the technical field of stem cells, particularly relates to a human mesenchymal stem cell factor, a preparation method and application thereof, and particularly relates to a method for preparing a stem cell active factor from umbilical cord mesenchymal stem cells. The method can prepare the stem cell active factor with high efficiency and stable performance.

Background

In recent years, with the intensive research on mesenchymal stem cells, secreted factors thereof have become hot spots of researchers. The mesenchymal stem cells can secrete various cell factors with biological activity, and the cell factors can effectively regulate and control the cell signal conduction of organisms and activate the human stem cells, so as to physiologically repair or replace cells with body injuries, pathological changes and aging.

The application of stem cells in cosmetology, which was the first application of stem cells in plastic surgery, is a stem cell cosmetology technology which is currently researched more. The stem cells used in plastic surgery are mainly human umbilical cord mesenchymal stem cells, human adipose mesenchymal stem cells and the like.

Traditionally, the so-called stem cell cosmetology is generally to inject stem cell injection directly. The source of the stem cells for injection is mainly extracted from aborted embryos, so that immunological rejection is inevitably caused, and certain theoretical problems exist. Even if autologous stem cells such as human adipose-derived mesenchymal stem cells are adopted, exogenous proteins (fetal bovine serum) are inevitably introduced in the culture and amplification process of the stem cells, and immune rejection reaction is easily caused. Therefore, scientists have recently proposed a new stem cell application method, the application of cell culture supernatant active factors and cell lysates.

The addition of the cytokines into the beauty cosmetics not only has the moisturizing and whitening effects of common cosmetics, but also can repair damaged skin, eliminate skin wrinkles, shrink pores, improve complexion and the like, and is more and more concerned by people.

Cytokine-containing cosmetics are now marketed in many countries, especially in korea, which is the most developed trade in cosmetic and plastic industries, and cytokine cosmetics are especially popular. In China, a plurality of large-scale stem cell companies develop cytokine cosmetics. However, the cytokine cosmetics in the market are generally manufactured by directly mixing the stem cell culture supernatant or mixing the stem cell culture supernatant into a cosmetic matrix after freeze-drying, the content of the cytokine is low, a large amount of sugar and other impurities are contained, and the discomfort such as skin dryness and the like can be caused after the cosmetics are used.

The research shows that the cell culture supernatant contains various cytokines with biological activity, and the application of the cell culture supernatant can avoid immunological rejection reaction generated during stem cell cosmetology. However, the liquid culture supernatant has a short storage time at room temperature, which hinders its popularization and application.

In addition, for the current stem cell sorting technology, magnetic bead sorting is common, but in the conventional sorting, the magnetic beads are subjected to subsequent culture along with the stem cells, that is, the magnetic beads are not separated from the stem cells, so that certain influence is certainly generated on the later production of the stem cells, and although some methods are adopted, such as adding some bacteria or other substances for degradation, other components such as exogenous proteins are added, so that secondary pollution to the stem cells is caused, and the problems are not actually solved.

At present, some reports on extraction and purification of stem cell culture supernatant exist, but the effect is still poor, for example, a patent with publication number CN102600057B discloses a preparation method of freeze-dried powder of human placental stem cell extract, wherein a single 3000D filter membrane is used for ultrafiltration and interception of the obtained cell culture solution, part of impurities are removed, but impurities such as saccharides and the like are not removed, and a large amount of loss of cytokines is caused, the repairing effect is poor, discomfort such as skin dryness and the like is caused, and the practical application value is low.

In addition, it is difficult to prepare stem cell active factors with high efficiency and/or stable performance.

Disclosure of Invention

The invention aims to provide a method for preparing stem cell active factors with high-efficiency and/or stable performance. It has been unexpectedly found that the beneficial effects of one or more aspects of the invention can be achieved and stem cell activating factors can be produced using the methods of the invention. The present invention has been completed based on this finding.

To this end, the present invention provides in a first aspect a method for preparing a stem cell activating factor, comprising the steps of:

(1) obtaining umbilical cord source mesenchymal stem cells:

taking umbilical cord of a healthy pregnant woman born by caesarean section at term, preparing the umbilical cord by a Wharton's Jelly separation method and a Wharton's Jelly suspension method; placing the Wharton jelly in a T75 culture bottle, adding a proper amount of Mesenchymal Stem Cell (MSC) serum-free culture medium, placing in a 5% CO2 incubator at 37 ℃ for culture, and culturing for 7 days to obtain cell colonies; the cells reach saturation about 10-13 days and can be passaged; the cells are in a long spindle shape, and are in a typical fibroblast-like shape;

(2) cell expansion:

when the cell fusion degree reaches 70-80%, gently washing the cells in the culture flask with physiological saline for 1-2 times, adding 3-5ml of digestive juice, standing at room temperature for 1-2 min, observing that the cells are approximately spherical under a mirror, gently tapping the wall of the flask, stopping digestion, transferring to a centrifuge tube, mixing, sampling and counting, wherein the cell fusion degree is 8000 cells/cm²Carrying out passage, repeating the above operations when the cell density reaches 70-80% in 2-3 days,to obtain sufficient quantities of MSCs, typically prepared using P3-P5 generation cells;

(3) cell identification and detection:

when the cell survival rate reaches more than 90%, the flow detection marker is qualified;

(4) preparation of stem cell active factor:

4i) when the cells are cultured to be the most vigorous, collecting the supernatant to a centrifuge tube, and filtering the supernatant by a 0.22 mu m filter membrane for sterilization;

4ii) cleaning the culture bottle with physiological saline for 2-3 times, discarding, adding 10ml of physiological saline/T225 culture bottle by adopting a cell starvation method, continuously culturing for 12-24 hours, blowing and uniformly mixing the cells until the cells become round, transferring the cells to a centrifuge tube, crushing the cells by adopting an ultrasonic crusher, centrifuging at 1400rpm for 5min, collecting supernatant, and filtering and sterilizing through a 0.22 mu m filter membrane;

4iii) mixing the supernatants obtained in the steps 4i) and 4ii), and concentrating by adopting a purification ultrafiltration system with the molecular weight cut-off of 10KD to obtain the stem cell active factor concentrated solution.

The method according to any one of the embodiments of the first aspect of the present invention, wherein the digestive juice in step (2) is 0.25% pancreatin.

The method according to any one of the embodiments of the first aspect of the present invention, wherein the flow detection marker in step (3) is: positive expression of CD73, CD90 and CD105, and negative expression of HLA-DR, CD11b, CD19, CD34 and CD 45.

The method according to any one of the embodiments of the first aspect of the present invention, wherein the flow assay marker results in step (3) indicate that the cells have adipogenic, osteogenic, chondrogenic differentiation potential.

The method according to any one of the embodiments of the first aspect of the present invention, wherein the culturing of the cells to the maximum in 4i) of step (4) means that the degree of cell fusion is about 70-80%.

The method according to any one of the embodiments of the first aspect of the present invention, wherein the step (4) of 4ii) comprises the steps of, when the cell is disrupted by the ultrasonicator: 4 ℃, 500w, sonication for 2 seconds, 8 seconds apart, 86 cycles.

The method according to any one of the embodiments of the first aspect of the present invention, further comprising the step of (5) freeze-drying the stem cell activating factor obtained in the step (4).

The method according to any one of the embodiments of the first aspect of the present invention, wherein the step of freeze-drying the stem cell activating factor (for long-term storage) comprises the following operations:

5i) adding a freeze-drying protective agent into the stem cell active factor concentrated solution obtained in the step (4), subpackaging the mixture into penicillin bottles, half plugging the vials, and placing the vials into a freeze dryer;

and 5ii) starting a freeze dryer, carrying out freeze drying on the sample according to a preset freeze drying program, and carrying out tamponade and sealing to obtain freeze-dried powder.

The freeze-dried powder thus obtained by freeze-drying usually has a loose porous tissue and can be stored at room temperature for a long period of time.

The method according to any one of the embodiments of the first aspect of the present invention, wherein said lyoprotectant in step 5i) of step (5) is selected from the group consisting of trehalose, mannitol, chitosan, dextran, glycine, arginine, glycine.

The method according to any of the embodiments of the first aspect of the present invention, wherein the concentration of the lyoprotectant in the concentrate in step (5) 5i) is between 0.5% and 10%, in particular between 1% and 6%.

The method according to any one of the embodiments of the first aspect of the present invention, wherein the dispensing in 5i) of step (5) is to add the stem cell activity factor concentrate into the vial in an amount of 3 ml/vial.

The method according to any one of the embodiments of the first aspect of the present invention, wherein the freeze-drying procedure in step (5) 5ii) comprises the following procedures:

pre-freezing: pre-freezing for 3.5h under normal pressure (the pre-freezing temperature is usually-30 to-40 ℃);

and (3) freeze drying a: starting a vacuum pump to ensure that the vacuum value is 0.014mbar, and freezing for 2h at the temperature of-38 to-40 ℃;

and (b) freeze drying: keeping the vacuum degree unchanged, heating to-20 to-25 ℃, and continuously freezing and drying for 12 hours under the condition; then heating to-2 ℃, and continuously drying for 2 hours under the condition;

and (3) resolving and drying: keeping the vacuum degree unchanged, heating to 33-37 ℃, and continuously drying for 2-5h under the condition.

The method according to any embodiment of the first aspect of the present invention, further comprising a step (6) of performing stem cell active factor detection on the stem cell active factor concentrated solution obtained in the step (4) or the stem cell active factor lyophilized powder obtained in the step (5), specifically as follows:

taking 3ml of stem cell active factor concentrated solution or stem cell active factor freeze-dried powder with the volume of 3ml before freeze-drying, diluting with 3ml of solvent (the solvent can be ultrapure water or hyaluronic acid solution), and determining the content of the stem cell active factor by adopting an enzyme linked immunosorbent assay kit coated by a recombinant human antibody (for example, the invention also adopts a commercialized kit provided by R & D company).

Further, the second aspect of the present invention provides a lyophilized powder of stem cell active factor, which comprises the stem cell active factor and a lyoprotectant.

The lyophilized powder of stem cell active factor according to any embodiment of the second aspect of the present invention, wherein the lyoprotectant is selected from the group consisting of trehalose, mannitol, chitosan, dextran, glycine, arginine, glycine.

The lyophilized powder of stem cell active factor according to any embodiment of the second aspect of the present invention is prepared substantially as follows:

(1) obtaining umbilical cord source mesenchymal stem cells:

taking umbilical cord of a healthy pregnant woman born by caesarean section at term, preparing the umbilical cord by a Wharton's Jelly separation method and a Wharton's Jelly suspension method; placing the Wharton jelly in a T75 culture bottle, adding a proper amount of Mesenchymal Stem Cell (MSC) serum-free culture medium, placing in a 5% CO2 incubator at 37 ℃ for culture, and culturing for 7 days to obtain cell colonies; the cells reach saturation about 10-13 days and can be passaged; the cells are in a long spindle shape, and are in a typical fibroblast-like shape;

(2) cell expansion:

when the cell fusion degree reaches 70-80%, gently washing the culture with physiological salineAdding 3-5ml of digestive juice into flask cell for 1-2 times, standing at room temperature for 1-2 min, observing under the mirror that the cell is nearly spherical, slightly beating the flask wall, stopping digestion, transferring to centrifuge tube, mixing, sampling, counting, and counting at 8000 cells/cm²Carrying out passage, repeating the above operations when the cell density reaches 70-80% in 2-3 days to obtain sufficient amount of MSC, and generally preparing by using P3-P5 generation cells;

(3) cell identification and detection:

when the cell survival rate reaches more than 90%, the flow detection marker is qualified;

(4) preparation of stem cell active factor:

4i) when the cells are cultured to be the most vigorous, collecting the supernatant to a centrifuge tube, and filtering the supernatant by a 0.22 mu m filter membrane for sterilization;

4ii) cleaning the culture bottle with physiological saline for 2-3 times, discarding, adding 10ml of physiological saline/T225 culture bottle by adopting a cell starvation method, continuously culturing for 12-24 hours, blowing and uniformly mixing the cells until the cells become round, transferring the cells to a centrifuge tube, crushing the cells by adopting an ultrasonic crusher, centrifuging at 1400rpm for 5min, collecting supernatant, and filtering and sterilizing through a 0.22 mu m filter membrane;

4iii) mixing the supernatants obtained in the steps 4i) and 4ii), and concentrating by adopting a purification ultrafiltration system with the molecular weight cut-off of 10KD to obtain a concentrated solution of the stem cell active factor;

(5) and (3) freeze drying:

5i) adding a freeze-drying protective agent into the stem cell active factor concentrated solution obtained in the step (4), subpackaging the mixture into penicillin bottles, half plugging the vials, and placing the vials into a freeze dryer;

and 5ii) starting a freeze dryer, carrying out freeze drying on the sample according to a preset freeze drying program, and carrying out tamponade and sealing to obtain freeze-dried powder.

The lyophilized powder of a stem cell active factor according to any one of the embodiments of the second aspect of the present invention, wherein the digestive juice in the step (2) is 0.25% pancreatin.

The lyophilized powder of stem cell active factor according to any embodiment of the second aspect of the present invention, wherein the flow detection marker in step (3) is: positive expression of CD73, CD90 and CD105, negative expression of HLA-DR, CD11b, CD19, CD34 and CD45

The lyophilized powder of stem cell active factor according to any embodiment of the second aspect of the present invention, wherein the flow assay marker result in step (3) shows that the cells have adipogenic, osteogenic, chondrogenic differentiation potential.

The lyophilized powder of stem cell active factor according to any one of the embodiments of the second aspect of the present invention, wherein the time when the cells are cultured to the maximum in 4i) of step (4) means that the degree of cell fusion is about 70-80%.

The lyophilized powder of stem cell active factor according to any embodiment of the second aspect of the present invention, wherein the procedure for disrupting the cells in step (4) 4ii) using the ultrasonicator is: 4 ℃, 500w, sonication for 2 seconds, 8 seconds apart, 86 cycles.

The lyophilized powder of stem cell active factor according to any embodiment of the second aspect of the present invention, wherein the lyoprotectant in step 5i) of step (5) is selected from trehalose, mannitol, chitosan, dextran, glycine, arginine, glycine.

The lyophilized powder of stem cell active factor according to any embodiment of the second aspect of the present invention, wherein the concentration of the lyoprotectant in the concentrate in 5i) of step (5) is 0.5% to 10%, particularly 1% to 6%.

The lyophilized powder of stem cell active factor according to any embodiment of the second aspect of the present invention, wherein the dispensing in 5i) of step (5) is to add the concentrated solution of stem cell active factor to the vial in an amount of 3 ml/vial.

The lyophilized powder of stem cell active factor according to any embodiment of the second aspect of the present invention, wherein the lyophilization process in step (5) 5ii) comprises the following procedures:

pre-freezing: pre-freezing for 3.5h under normal pressure (the pre-freezing temperature is usually-30 to-40 ℃);

and (3) freeze drying a: starting a vacuum pump to ensure that the vacuum value is 0.014mbar, and freezing for 2h at the temperature of-38 to-40 ℃;

and (b) freeze drying: keeping the vacuum degree unchanged, heating to-20 to-25 ℃, and continuously freezing and drying for 12 hours under the condition; then heating to-2 ℃, and continuously drying for 2 hours under the condition;

and (3) resolving and drying: keeping the vacuum degree unchanged, heating to 33-37 ℃, and continuously drying for 2-5h under the condition.

The lyophilized powder of stem cell active factor according to any embodiment of the second aspect of the present invention, which is prepared by further comprising step 6), is to perform the operation of stem cell active factor detection on the concentrated solution of stem cell active factor obtained in step (4) or the lyophilized powder of stem cell active factor obtained in step (5), and specifically comprises the following steps:

taking 3ml of stem cell active factor concentrated solution or stem cell active factor freeze-dried powder with the volume of 3ml before freeze-drying, diluting with 3ml of solvent (the solvent can be ultrapure water or hyaluronic acid solution), and determining the content of the stem cell active factor by adopting an enzyme linked immunosorbent assay kit coated by a recombinant human antibody (for example, the invention also adopts a commercialized kit provided by R & D company).

It has been found that when sodium citrate is added to a physiological saline solution at a concentration of 0.1 to 0.2mmol/L during further culturing in a 10ml physiological saline/T225 flask by cell starvation in step 4ii) of the present invention for 12 to 24 hours (this sodium citrate is introduced into the subsequent ultrafiltration concentration step), after the ultrafiltration concentration in step 4iii), the content of active factors in the concentrate (expressed as the total of four typical active factors EGF, FGF, VEGF, and IL-6, the same applies; expressed as C1) and the content of active factors in the concentrate (expressed as C2) are determined, and the percent discard is calculated as follows, wherein the percent discard is (C2/C1) ×% when the percent discard is lower, the loss of active factors is smaller, the results show that in each example of the following invention for preparing a concentrated solution of dry cell active factors, when sodium citrate is added at a concentration of 0.1 to 0.2mmol/L in the above step 4ii), the percent discard is less than 1.7% when the percent discard is 1.7% and the percent discard is 1.7.7.7% is calculated as the percent discard of the total of the percent discard of sodium citrate added in the above step 4ml physiological saline, the above example, the percent discard is 1.6, and the percent discard is calculated as the percent discard of the concentration of the above example 7.7.7.6, the above example 7.7.7.7.7% when the percent discard is calculated as the concentration step 4 to 1.6, the percent of the concentration of the above example, the concentration step 4ml physiological saline added

Any technical feature possessed by any one aspect of the invention or any embodiment of that aspect is equally applicable to any other embodiment or any embodiment of any other aspect, so long as they are not mutually inconsistent, although appropriate modifications to the respective features may be made as necessary when applicable to each other. Various aspects and features of the disclosure are described further below.

All documents cited herein are incorporated by reference in their entirety and to the extent such documents do not conform to the meaning of the present invention, the present invention shall control. Further, the various terms and phrases used herein have the ordinary meaning as is known to those skilled in the art, and even though such terms and phrases are intended to be described or explained in greater detail herein, reference is made to the term and phrase as being inconsistent with the known meaning and meaning as is accorded to such meaning throughout this disclosure.

The stem cell active factor or the freeze-dried powder thereof can be directly matched with a solvent (the solvent can be ultrapure water or a hyaluronic acid solution), and can be directly used for beautifying and protecting skin, including beautifying, removing freckles, repairing scars and the like; or the stem cell active factor or the freeze-dried powder thereof obtained by the invention can be directly added into beauty and skin care products of various preparations for use; or the stem cell active factor or the freeze-dried powder thereof obtained by the invention can be directly used as an injection, or used as a cream, an emulsion, a water solution or various common preparations.

The method of the invention has the following beneficial effects:

1. the cell source has homogeneity and accords with the biological characteristics of stem cells, thereby ensuring the secretion amount and performance stability of stem cell source active factors;

2. the culture medium does not contain sensitizing components such as antibiotics and fetal calf serum, and is suitable for various people (especially people with streptomycin allergy).

3. The lyophilized powder preparation is convenient for long-distance transportation and long-term storage.

Drawings

FIG.1P 0 generation cytogram (× 4)

FIG. 2P 5 generation cytogram (× 10)

FIG. 3: and (3) detection result of cell flow markers in P5 generation.

FIG. 4: the P5 generation stem cell differentiation potential test shows the test results of the control group, the chondrogenic group, the adipogenic group and the osteogenic group.

Detailed Description

The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention. The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible.

Example 1: preparing concentrated solution of stem cell active factor

(1) Obtaining umbilical cord source mesenchymal stem cells:

taking umbilical cord of a healthy pregnant woman born by caesarean section at term, preparing the umbilical cord by a Wharton's Jelly separation method and a Wharton's Jelly suspension method; placing the Wharton jelly in a T75 culture bottle, adding a proper amount of Mesenchymal Stem Cell (MSC) serum-free culture medium, placing in a 5% CO2 incubator at 37 ℃ for culture, and culturing for 7 days to obtain cell colonies; the cells reach saturation about 12 days and can be passaged; the cells are in the form of long spindle, typical of fibroblast-like morphology (see, e.g., fig. 1);

(2) cell expansion:

when the cell fusion degree reaches 70-80%, gently washing the cells in the culture flask with physiological saline for 1-2 times, adding 4ml of digestive juice (0.25% pancreatin), standing at room temperature for 1-2 min, observing the cells to be close to spherical under the microscope, gently tapping the flask wall, stopping digestion, transferring to a place where the cells are fused, and collecting the cellsMixing the materials in a centrifuge tube, sampling, counting, and processing at 8000 cells/cm²Passaging is carried out, and when the cell density reaches 70-80% in 2-3 days, the above operations are repeated to obtain sufficient amount of MSC, and the MSC is generally prepared by using P3-P5 generation cells (in this case, P5 generation cells are used, and a micrograph thereof is shown in FIG. 2, for example);

(3) cell identification and detection:

when the cell viability reaches more than 90%, the flow detection marker is qualified, and the standard is as follows: CD73, CD90, CD105 positive, HLA-DR, CD11b, CD19, CD34, CD45 negative (see, e.g., fig. 3), and the results show that the cells have adipogenic, osteogenic, chondrogenic differentiation potential (see, e.g., fig. 4);

(4) preparation of stem cell active factor:

4i) when the cells are cultured to be the most vigorous (namely the cell fusion degree is about 70-80%), collecting the supernatant to a centrifuge tube, and filtering the supernatant by a 0.22 mu m filter membrane for sterilization;

4ii) washing the culture bottle with physiological saline for 2-3 times, discarding, adding 10ml of physiological saline/T225 culture bottle by adopting a cell starvation method, continuously culturing for 18 hours, moving to a centrifuge tube after the cells are rounded and uniformly blown, and crushing the cells by adopting an ultrasonic crusher (the procedure is as follows: 4 ℃, 500w, 2 seconds of ultrasound, 8 seconds of interval, 86 cycles), then centrifuging at 1400rpm for 5min, collecting the supernatant, and filtering and sterilizing through a 0.22 mu m filter membrane;

4iii) mixing the supernatants obtained in the steps 4i) and 4ii), and concentrating by adopting a purification ultrafiltration system with the molecular weight cut-off of 10KD to obtain the stem cell active factor concentrated solution.

And (3) measuring an active factor: taking 3ml of the stem cell active factor concentrated solution, determining the content of stem cell active factors (four typical active factors, namely EGF, FGF, VEGF and IL-6, are determined) by adopting an enzyme linked immunosorbent assay kit (supplied by R & D company) coated by a recombinant humanized antibody, and taking the total amount of the four typical active factors as the total active factor content of the concentrated solution. In addition, the total active factor content in the waste liquid obtained in step 4iii) is determined in the same manner.

In addition, the recovery rates of the four typical active factors of EGF, FGF, VEGF and IL-6 are respectively calculated, and the results show that the recovery rates of the EGF, the FGF, the VEGF and the IL-6 in the method (the embodiment 1 to the embodiment 3) are respectively more than 92.2%, more than 94.1%, 91.6% and 92.7%, which indicates that the method has excellent recovery rates of the active factors. When the active factors are prepared by the methods described in CN105543313A (201511016742.4) specifications [0037] to [0060], the recovery rate of IL-6 is measured to be less than 87%.

Example 2: preparing concentrated solution of stem cell active factor

(1) Obtaining umbilical cord source mesenchymal stem cells:

taking umbilical cord of a healthy pregnant woman born by caesarean section at term, preparing the umbilical cord by a Wharton's Jelly separation method and a Wharton's Jelly suspension method; placing the Wharton jelly in a T75 culture bottle, adding a proper amount of Mesenchymal Stem Cell (MSC) serum-free culture medium, placing in a 5% CO2 incubator at 37 ℃ for culture, and culturing for 7 days to obtain cell colonies; the cells reach saturation in about 10 days and can be passaged; the cells are in a long spindle shape, and are in a typical fibroblast-like shape;

(2) cell expansion:

when the cell fusion degree reaches 70-80%, gently washing the cells in the culture flask with physiological saline for 1-2 times, adding 5ml of digestive juice (0.25% pancreatin), standing at room temperature for 1-2 min, observing the cells to be approximately spherical under the microscope, gently tapping the flask wall, stopping digestion, transferring to a centrifuge tube, mixing, sampling, counting, and counting at 8000 cells/cm²Carrying out passage, repeating the above operations when the cell density reaches 70-80% in 2-3 days to obtain sufficient amount of MSC, and generally preparing by using P4 generation cells;

(3) cell identification and detection:

when the cell viability reaches more than 90%, the flow detection marker is qualified, and the standard is as follows: CD73, CD90 and CD105 are positive in expression, HLA-DR, CD11b, CD19, CD34 and CD45 are negative in expression, and the results show that the cells have adipogenic, osteogenic and chondrogenic differentiation potentials;

(4) preparation of stem cell active factor:

4i) when the cells are cultured to be the most vigorous (namely the cell fusion degree is about 70-80%), collecting the supernatant to a centrifuge tube, and filtering the supernatant by a 0.22 mu m filter membrane for sterilization;

4ii) washing the culture bottle with physiological saline for 2-3 times, discarding, adding 10ml of physiological saline/T225 culture bottle by adopting a cell starvation method, continuously culturing for 12 hours, moving to a centrifuge tube after the cells are rounded and uniformly blown, and crushing the cells by adopting an ultrasonic crusher (the procedure is as follows: 4 ℃, 500w, 2 seconds of ultrasound, 8 seconds of interval, 86 cycles), then centrifuging at 1400rpm for 5min, collecting the supernatant, and filtering and sterilizing through a 0.22 mu m filter membrane;

4iii) mixing the supernatants obtained in the steps 4i) and 4ii), and concentrating by adopting a purification ultrafiltration system with the molecular weight cut-off of 10KD to obtain the stem cell active factor concentrated solution.

And (3) measuring an active factor: taking 3ml of the stem cell active factor concentrated solution, determining the content of stem cell active factors (four typical active factors, namely EGF, FGF, VEGF and IL-6, are determined) by adopting an enzyme linked immunosorbent assay kit (supplied by R & D company) coated by a recombinant humanized antibody, and taking the total amount of the four typical active factors as the total active factor content of the concentrated solution. In addition, the total active factor content in the waste liquid obtained in step 4iii) is determined in the same manner.

Example 3: preparing concentrated solution of stem cell active factor

(1) Obtaining umbilical cord source mesenchymal stem cells:

taking umbilical cord of a healthy pregnant woman born by caesarean section at term, preparing the umbilical cord by a Wharton's Jelly separation method and a Wharton's Jelly suspension method; placing the Wharton jelly in a T75 culture bottle, adding a proper amount of Mesenchymal Stem Cell (MSC) serum-free culture medium, placing in a 5% CO2 incubator at 37 ℃ for culture, and culturing for 7 days to obtain cell colonies; the cells reach saturation about 13 days and can be passaged; the cells are in a long spindle shape, and are in a typical fibroblast-like shape;

(2) cell expansion:

when the cell fusion degree reaches 70-80%, gently washing the cells in the culture flask with physiological saline for 1-2 times, adding 3ml of digestive juice (0.25% pancreatin), standing at room temperature for 1-2 min, observing the cells to be approximately spherical under the microscope, gently tapping the flask wall, stopping digestion, transferring to a centrifuge tube, mixing, sampling, counting, and counting at 8000 cells/cm²Carrying out passage, repeating the above operations when the cell density reaches 70-80% in 2-3 days to obtain sufficient amount of MSC, and generally preparing by using P3 generation cells;

(3) cell identification and detection:

when the cell viability reaches more than 90%, the flow detection marker is qualified, and the standard is as follows: CD73, CD90 and CD105 are positive in expression, HLA-DR, CD11b, CD19, CD34 and CD45 are negative in expression, and the results show that the cells have adipogenic, osteogenic and chondrogenic differentiation potentials;

(4) preparation of stem cell active factor:

4i) when the cells are cultured to be the most vigorous (namely the cell fusion degree is about 70-80%), collecting the supernatant to a centrifuge tube, and filtering the supernatant by a 0.22 mu m filter membrane for sterilization;

4ii) washing the culture bottle with physiological saline for 2-3 times, discarding, adding 10ml of physiological saline/T225 culture bottle by adopting a cell starvation method, continuously culturing for 24 hours, moving to a centrifuge tube after the cells are rounded and uniformly blown, and crushing the cells by adopting an ultrasonic crusher (the procedure is as follows: 4 ℃, 500w, 2 seconds of ultrasound, 8 seconds of interval, 86 cycles), then centrifuging at 1400rpm for 5min, collecting the supernatant, and filtering and sterilizing through a 0.22 mu m filter membrane;

4iii) mixing the supernatants obtained in the steps 4i) and 4ii), and concentrating by adopting a purification ultrafiltration system with the molecular weight cut-off of 10KD to obtain the stem cell active factor concentrated solution.

And (3) measuring an active factor: taking 3ml of the stem cell active factor concentrated solution, determining the content of stem cell active factors (four typical active factors, namely EGF, FGF, VEGF and IL-6, are determined) by adopting an enzyme linked immunosorbent assay kit (supplied by R & D company) coated by a recombinant humanized antibody, and taking the total amount of the four typical active factors as the total active factor content of the concentrated solution. In addition, the total active factor content in the waste liquid obtained in step 4iii) is determined in the same manner.

Lyophilized powder example 1: preparing lyophilized powder of stem cell active factor

5i) Taking the stem cell active factor concentrated solution obtained in the step 1, adding a freeze-drying protective agent (trehalose) into the concentrated solution, dissolving the freeze-drying protective agent until the concentration of the freeze-drying protective agent dissolved in the freeze-drying protective agent reaches 4%, subpackaging the obtained mixture into penicillin bottles (3 ml/piece), half plugging the penicillin bottles, and placing the penicillin bottles into a freeze dryer;

5ii) starting a freeze dryer, carrying out freeze drying on the sample according to a preset freeze drying program, and carrying out tamponade sealing to obtain freeze-dried powder; the freeze-drying procedure was as follows:

pre-freezing: pre-freezing for 3.5h (the pre-freezing temperature is-35 ℃) under normal pressure;

and (3) freeze drying a: starting a vacuum pump to ensure that the vacuum value is 0.014mbar, and freezing for 2h at the temperature of-38 to-40 ℃;

and (b) freeze drying: maintaining the vacuum degree unchanged, heating to-23 ℃, and continuously freezing and drying for 12 hours under the condition; then heating to-2 ℃, and continuously drying for 2 hours under the condition;

and (3) resolving and drying: keeping the vacuum degree unchanged, heating to 35 ℃, and continuing drying for 4 hours under the condition.

And (3) determining the active factors of the freeze-dried powder:

the results show that the total active factor content of all the freeze-dried powders of freeze-dried powder examples 1 to 5 is within the range of 98 to 101 percent, and the freeze-dried powder of the invention has excellent properties and no loss of active factors in the process of preparing the freeze-dried powder.

Lyophilized powder example 2: preparing lyophilized powder of stem cell active factor

5i) Taking the stem cell active factor concentrated solution obtained in the step 2, adding a freeze-drying protective agent (mannitol) into the concentrated solution, dissolving the freeze-drying protective agent until the concentration of the dissolved freeze-drying protective agent reaches 1%, subpackaging the obtained mixture into penicillin bottles (3 ml/piece), half plugging the penicillin bottles, and placing the penicillin bottles into a freeze dryer;

5ii) starting a freeze dryer, carrying out freeze drying on the sample according to a preset freeze drying program, and carrying out tamponade sealing to obtain freeze-dried powder; the freeze-drying procedure was as follows:

pre-freezing: pre-freezing for 3.5h under normal pressure (the pre-freezing temperature is-40 ℃);

and (3) freeze drying a: starting a vacuum pump to ensure that the vacuum value is 0.014mbar, and freezing for 2h at the temperature of-38 to-40 ℃;

and (b) freeze drying: maintaining the vacuum degree, heating to-20 deg.C, and freeze drying for 12 hr; then heating to-2 ℃, and continuously drying for 2 hours under the condition;

and (3) resolving and drying: keeping the vacuum degree unchanged, heating to 37 ℃, and continuing drying for 2h under the condition.

Lyophilized powder example 3: preparing lyophilized powder of stem cell active factor

5i) Taking the stem cell active factor concentrated solution obtained in the step 3, adding a freeze-drying protective agent (chitosan) into the concentrated solution, dissolving the freeze-drying protective agent until the concentration of the freeze-drying protective agent dissolved in the freeze-drying protective agent reaches 6%, subpackaging the mixture into penicillin bottles (3 ml/piece), half plugging the bottles, and placing the bottles into a freeze dryer;

5ii) starting a freeze dryer, carrying out freeze drying on the sample according to a preset freeze drying program, and carrying out tamponade sealing to obtain freeze-dried powder; the freeze-drying procedure was as follows:

pre-freezing: pre-freezing for 3.5h (the pre-freezing temperature is-30 ℃) under normal pressure;

and (3) freeze drying a: starting a vacuum pump to ensure that the vacuum value is 0.014mbar, and freezing for 2h at the temperature of-38 to-40 ℃;

and (b) freeze drying: maintaining the vacuum degree unchanged, heating to-25 ℃, and continuously freezing and drying for 12 hours under the condition; then heating to-2 ℃, and continuously drying for 2 hours under the condition;

and (3) resolving and drying: maintaining the vacuum degree, heating to 33 ℃, and continuing drying for 5h under the condition.

Lyophilized powder example 4: preparing lyophilized powder of stem cell active factor

5i) Respectively taking the stem cell active factor concentrated solutions obtained in the steps of examples 1, 2 and 3, adding a freeze-drying protective agent (dextran) into the concentrated solutions, dissolving the freeze-drying protective agent until the concentration of the freeze-drying protective agent after being dissolved in the concentrated solutions reaches 4%, subpackaging the obtained mixture into penicillin bottles (3 ml/piece), half plugging the penicillin bottles, and placing the obtained product into a freeze dryer;

5ii) starting a freeze dryer, carrying out freeze drying on the sample according to a preset freeze drying program, and carrying out tamponade sealing to obtain freeze-dried powder; the freeze-drying procedure was as follows:

pre-freezing: pre-freezing for 3.5h (the pre-freezing temperature is-35 ℃) under normal pressure;

and (3) freeze drying a: starting a vacuum pump to ensure that the vacuum value is 0.014mbar, and freezing for 2h at the temperature of-38 to-40 ℃;

and (b) freeze drying: maintaining the vacuum degree unchanged, heating to-25 ℃, and continuously freezing and drying for 12 hours under the condition; then heating to-2 ℃, and continuously drying for 2 hours under the condition;

and (3) resolving and drying: keeping the vacuum degree unchanged, heating to 35 ℃, and continuing drying for 3 hours under the condition.

Lyophilized powder example 5: preparing lyophilized powder of stem cell active factor

5i) Respectively taking the stem cell active factor concentrated solutions obtained in the steps of examples 1, 2 and 3, adding a freeze-drying protective agent (glycine) into the concentrated solutions, dissolving the freeze-drying protective agent (glycine) until the concentration of the freeze-drying protective agent in the concentrated solutions reaches 3%, subpackaging the obtained mixture into penicillin bottles (3 ml/piece), half plugging the penicillin bottles, and placing the penicillin bottles into a freeze dryer;

5ii) starting a freeze dryer, carrying out freeze drying on the sample according to a preset freeze drying program, and carrying out tamponade sealing to obtain freeze-dried powder; the freeze-drying procedure was as follows:

pre-freezing: pre-freezing for 3.5h under normal pressure (the pre-freezing temperature is-37 ℃);

and (3) freeze drying a: starting a vacuum pump to ensure that the vacuum value is 0.014mbar, and freezing for 2h at the temperature of-38 to-40 ℃;

and (b) freeze drying: maintaining the vacuum degree unchanged, heating to-25 ℃, and continuously freezing and drying for 12 hours under the condition; then heating to-2 ℃, and continuously drying for 2 hours under the condition;

and (3) resolving and drying: keeping the vacuum degree unchanged, heating to 35 ℃, and continuing drying for 4 hours under the condition.

Claims (9)

1. A method of preparing a stem cell activating factor comprising the steps of:

(1) obtaining umbilical cord source mesenchymal stem cells:

taking umbilical cord of a healthy pregnant woman born by caesarean section at full term, and preparing by a separation Wharton's jelly method and a Wharton's jelly suspension method; putting the Wharton jelly into a T75 culture bottle, adding a proper amount of mesenchymal stem cell serum-free culture medium, placing the bottle in a 5% CO2 incubator at 37 ℃ for culture, and culturing for 7 days to obtain cell colonies; after 10-13 days, the cells are saturated and passage is carried out;

(2) cell expansion:

when the cell fusion degree reaches 70-80%, gently washing the cells in the culture flask with physiological saline for 1-2 times, adding 3-5ml of digestive juice, standing at room temperature for 1-2 min, observing that the cells are approximately spherical under a mirror, gently tapping the wall of the flask, stopping digestion, transferring to a centrifuge tube, mixing, sampling and counting, wherein the cell fusion degree is 8000 cells/cm²Carrying out passage, repeating the above operations when the cell density reaches 70-80% in 2-3 days to obtain a sufficient amount of mesenchymal stem cells, and preparing by adopting P3-P5 generation cells;

(3) cell identification and detection:

when the cell survival rate reaches more than 90%, the flow detection marker is qualified;

(4) preparation of stem cell active factor:

4i) when the cells are cultured to be the most vigorous, collecting the supernatant to a centrifuge tube, and filtering the supernatant by a 0.22 mu m filter membrane for sterilization; wherein the cell culture to the maximum is 70-80% of cell fusion degree;

4ii) cleaning the culture bottle with physiological saline for 2-3 times, discarding, adding 10ml of physiological saline/T225 culture bottle by adopting a cell starvation method, continuously culturing for 12-24 hours, blowing and uniformly mixing the cells until the cells become round, transferring the cells to a centrifuge tube, crushing the cells by adopting an ultrasonic crusher, centrifuging at 1400rpm for 5min, collecting supernatant, and filtering and sterilizing through a 0.22 mu m filter membrane; wherein sodium citrate with the concentration of 0.1-0.2 mmol/L is added into the physiological saline;

4iii) mixing the supernatants obtained in the steps 4i) and 4ii), and concentrating by adopting a purification ultrafiltration system with the molecular weight cut-off of 10KD to obtain a concentrated solution of the stem cell active factor; and

(5) further carrying out freeze drying on the stem cell active factor obtained in the step (4), wherein the freeze drying comprises the following operation steps:

5i) adding a freeze-drying protective agent into the stem cell active factor concentrated solution obtained in the step (4), subpackaging the mixture into penicillin bottles, half plugging the vials, and placing the vials into a freeze dryer; the freeze-drying protective agent is selected from trehalose, mannitol, chitosan, dextran, glycine, arginine and aminoacetic acid, and the concentration of the freeze-drying protective agent in the concentrated solution is 1% -6%;

and 5ii) starting a freeze dryer, carrying out freeze drying on the sample according to a preset freeze drying program, and carrying out tamponade and sealing to obtain freeze-dried powder.

2. The method according to claim 1, wherein the digestive juice in step (2) is 0.25% pancreatin.

3. The method according to claim 1, wherein the flow detection marker in step (3) is: positive expression of CD73, CD90 and CD105, and negative expression of HLA-DR, CD11b, CD19, CD34 and CD 45.

4. The method according to claim 1, wherein the flow assay marker results in step (3) indicate that the cells have adipogenic, osteogenic, chondrogenic differentiation potential.

5. The method according to claim 1, wherein the step (4) of (ii) comprises the steps of, when the cell is disrupted by the ultrasonicator: 4 ℃, 500w, sonication for 2 seconds, 8 seconds apart, 86 cycles.

6. The method according to claim 1, wherein the dispensing in step (5) 5i) is performed by adding the concentrated solution of the stem cell activating factor to the vial in an amount of 3 ml/vial.

7. The method according to claim 1, wherein said freeze-drying procedure of step (5) 5ii) comprises the following operating procedures:

pre-freezing: pre-freezing for 3.5 hours under normal pressure, wherein the pre-freezing temperature is-30 to-40 ℃;

and (3) freeze drying a: starting a vacuum pump to ensure that the vacuum value is 0.014mbar, and freezing for 2h at the temperature of-38 to-40 ℃;

and (b) freeze drying: keeping the vacuum degree unchanged, heating to-20 to-25 ℃, and continuously freezing and drying for 12 hours under the condition; then heating to-2 ℃, and continuously drying for 2h under the condition;

and (3) resolving and drying: keeping the vacuum degree unchanged, heating to 33-37 ℃, and continuously drying for 2-5h under the condition.

8. The method according to claim 1, further comprising a step (6) of performing stem cell active factor detection on the stem cell active factor concentrated solution obtained in the step (4) or the stem cell active factor lyophilized powder obtained in the step (5), specifically as follows:

taking 3ml of stem cell active factor concentrated solution or 3ml of stem cell active factor freeze-dried powder with volume before freeze-drying, diluting with 3ml of solvent, and determining the content of the stem cell active factor by adopting an enzyme linked immunosorbent assay kit coated by the recombinant humanized antibody.

9. The method according to claim 8, wherein the solvent is ultrapure water or a hyaluronic acid solution.

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