CN114196626A

CN114196626A - Umbilical cord mesenchymal stem cells and isolated culture and amplification method thereof

Info

Publication number: CN114196626A
Application number: CN202210139582.6A
Authority: CN
Inventors: 李晓玉; 杨治权; 李若鲲; 武威; 马浩天; 陈晖�; 李慧亮
Original assignee: Beijing Guowei Biotechnology Co ltd
Current assignee: Beijing Guowei Biotechnology Co ltd
Priority date: 2022-02-16
Filing date: 2022-02-16
Publication date: 2022-03-18

Abstract

The invention discloses umbilical cord mesenchymal stem cells and a separation, culture and amplification method thereof. The separation, culture and amplification method of the umbilical cord mesenchymal stem cells can enable the Wharton's jelly to be processed into smaller tissue blocks, thereby being more beneficial to the separation of the stem cells. Meanwhile, the adverse effect on stem cells in the tissue caused by excessive breakage of the tissue block is reduced by adding adenine serving as a protective component, and the tissue block is prevented from floating in the culture medium to be unfavorable for adhering to the bottom of a culture container through standing treatment and a specific adherent culture medium, so that the defects of climbing out and separation of the stem cells are not favorable.

Description

Umbilical cord mesenchymal stem cells and isolated culture and amplification method thereof

Technical Field

The invention relates to stem cell separation, in particular to a method for separating or further culturing mesenchymal stem cells in umbilical cord Wharton's jelly and umbilical cord mesenchymal stem cells obtained by the method.

Background

Mesenchymal stem cells refer to a class of adult stem cells with self-renewal and multipotentiality, and are mainly derived from mesoderm tissues. Since the first discovery and successful isolation of MSCs in bone marrow in the last 60 th century, subsequent researchers isolated MSCs from tissues such as fat, placenta, umbilical cord, dental pulp, and the like. MSCs are capable of differentiating into multiple blastoderm multi-type cells, such as epiblast-like cells of the ectoderm; adipocytes, chondrocytes, osteocytes of mesoderm, etc.; endodermal insulin-secreting cells, and the like. The MSC has various sources, easy separation, amplification and culture, low immunogenicity, strong updating and differentiation capacity, wide treatment range and the like.

The umbilical cord is one of the isolated sources of mesenchymal stem cells, and the isolation, expansion and culture of umbilical cord mesenchymal stem cells from human umbilical cord Wharton's jelly is an important mode. Currently, the isolation culture methods commonly used for such stem cells are enzyme digestion and tissue block adherent culture. The enzyme digestion method can obtain a large amount of primary cells, but the damage of digestive enzyme in the extraction process causes poor cell adherence effect and form, the digestion time of tissue blocks is difficult to be uniformly quantified in the large-scale production process, in addition, the separation and extraction cost is high, and the processing time is relatively long. The traditional tissue block culture method has the defect that the primary culture time is generally long and needs about 15 days.

The information in this background is only for the purpose of illustrating the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art that is known to a person skilled in the art.

Disclosure of Invention

In order to solve at least part of technical problems in the prior art, the invention provides a separation culture amplification method of umbilical cord mesenchymal stem cells. The method can reduce the damage of the enzyme digestion method to the primary cells and avoid the defect of long time consumption of the tissue block culture method. Specifically, the present invention includes the following.

In a first aspect of the present invention, there is provided a method for isolated culture and expansion of umbilical cord mesenchymal stem cells, comprising the following steps:

(1)dividing the washed Wharton's jelly into 0.5-1.0mm³Centrifugally collecting the small tissues, flatly paving the small tissues on a culture substrate, and standing for 10-24 hours at the temperature of 30-40 ℃ and the humidity of 100%;

(2) slowly adding an adherent culture medium which is 3-5 times the volume of the small tissue into the small tissue after standing, wherein the adherent culture medium comprises 5-15 parts by volume of serum substitute, 80-95 parts by volume of alpha-MEM/DMEM-F12, 5-15ng/mL of b-FGF, 0.5-1.5% adenine and 2-3mg/mL dopamine;

(3) removing the adherent culture medium, adding the primary culture medium, and continuing culturing until the umbilical cord mesenchymal stem cells are moved out to the periphery of the small tissue; and

(4) collecting primary cells for subculturing, and collecting subculturing cells to obtain the umbilical cord mesenchymal stem cells.

According to the method for separating, culturing and amplifying the umbilical cord mesenchymal stem cells, preferably, the primary cell culture medium comprises 0.05-0.2 volume part of beta-mercaptoethanol, 0.5-2 volume parts of non-essential amino acid aqueous solution, 5-15 volume parts of serum substitute, 80-95 volume parts of alpha-MEM/DMEM-F12, 5-15ng/mL of b-FGF, 0.5-1.5% adenine and 0.2-1mg/mL dopamine.

According to the method for separating, culturing and amplifying the umbilical cord mesenchymal stem cells, preferably, after the small pieces of tissue are collected by centrifugation, the small pieces of tissue are laid on a culture substrate without adding a culture medium or liquid.

According to the method for separating, culturing and amplifying the umbilical cord mesenchymal stem cells, preferably, when the fusion degree of the primary cells reaches more than 80%, the primary cells are collected by treating the cells with digestive enzyme.

According to the method for separating, culturing and amplifying the umbilical cord mesenchymal stem cells, preferably, the mesenchymal stem cells highly express CD73, CD90 and CD105, and simultaneously, lowly express or do not express CD34, CD45, HLA-DR, CD11b and CD 19.

According to the method for separating, culturing and amplifying the umbilical cord mesenchymal stem cells, preferably, the adherent culture medium is weakly alkaline at least when the umbilical cord mesenchymal stem cells are initially contacted with the tissue block.

According to the method for the isolated culture and amplification of the umbilical cord mesenchymal stem cells, preferably, the weak alkaline is pH in the range of 7.5-7.8.

According to the method for separating, culturing and amplifying the umbilical cord mesenchymal stem cells, preferably, the Wharton's jelly is washed by using a washing solution, wherein the washing solution is a physiological saline solution containing penicillin, streptomycin and amphotericin.

According to the method for separating, culturing and amplifying the umbilical cord mesenchymal stem cells, preferably, the serum-free culture medium is free of animal-derived components and human platelet lysate.

In a second aspect of the present invention, there is provided an umbilical cord mesenchymal stem cell prepared by the amplification method of the first aspect.

The method for separating, culturing and amplifying the umbilical cord mesenchymal stem cells can treat the Wharton's jelly into smaller tissue blocks which reach 1mm³Hereinafter, preferably 0.2 to 1mm³E.g. 0.5-0.8mm³Thereby facilitating the separation of stem cells.

Meanwhile, the invention reduces the adverse effect on stem cells in the tissue caused by excessive breakage of tissue blocks by adding adenine as a protective component. On the other hand, the tissue blocks are prevented from floating in the culture medium through standing treatment and a specific adherence culture medium, so that the defects that the tissue blocks are not favorable for adhering to the bottom of the culture container and further are not favorable for climbing out and separating stem cells are overcome.

Drawings

FIG. 1 shows the climbing of dry cells under different conditions. Wherein A is 2mm³Directly adding primary culture medium to the left and right tissue blocks to culture for 5 days; b is 0.5mm³Left and right tissue blocks are subjected to standing treatment and then are not subjected to adherent culture for 5 days; c is 0.5mm³Standing the left tissue block and the right tissue block, and then performing adherent culture for 5 days to obtain a result graph; d is 0.2mm³Left and right tissue blocks are subjected to standing treatment, and then result graphs of adherent culture are carried out for 5 days.

FIG. 2 shows the flow cytometry results of the cells obtained by separation.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that the upper and lower limits of the range, and each intervening value therebetween, is specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control. Unless otherwise indicated, "%" is percent by weight.

Herein, the term "Wharton's jelly" means a gelatinous material constituting the umbilical cord, the main component of which is mucopolysaccharide, a mucosal tissue containing active cells such as fibroblasts, macrophages, and a small amount of cells having stem cell characteristics, particularly umbilical cord mesenchymal stem cells (huc-MSCs) are abundant.

Herein, the term "umbilical cord mesenchymal stem cell" refers to a multifunctional stem cell existing in umbilical cord tissue of newborn, which can be differentiated into a variety of tissue cells, and has a wide clinical application prospect. The umbilical cord mesenchymal stem cells are stem cells separated from Wharton's jelly, particularly human Wharton's jelly. The flow cytometry detection shows that the umbilical cord mesenchymal stem cells of the invention highly express CD73, CD90 and CD105, and simultaneously, lowly express or do not express CD34, CD45, HLA-DR, CD11b and CD 19.

Herein, the term "small tissue" is sometimes simply referred to as "tissue mass of the present invention" and refers to a massive tissue obtained by physically dividing the tissue by the Wharton's jelly, which is most advantageous for the stem cells to climb out of the tissue mass while avoiding physical damage to the stem cells in the tissue as much as possible. The volume of the tissue block used in the conventional adherent culture of the tissue block in the field is generally more than 1.0mm³E.g. 3.0mm³Even 5.0mm³The above. Typically, the tissue mass of the present invention has a volume of 1.0mm³This is to be distinguished from the conventional tissue adherent culture isolation method in the art. Unlike conventional tissue block adherent culture isolation methods of the art, the present invention enables the use of smaller volumes of tissue blocks while reducing or avoiding adverse effects resulting from excessive disruption of tissue. On the other hand, the tissue mass of the present invention preferably has a volume larger than that of the tissue mass in the enzymatic digestion method. Thus, preferably, the tissue mass of the present invention has a volume generally in the range of 0.2 to 1.0mm³Preferably 0.2 to 0.8mm³E.g. 0.4mm³Left and right, 0.5mm³Left and right, 0.6mm³Left and right, 0.7mm³Left and right, etc.

Isolation culture amplification method

In a first aspect of the present invention, there is provided an isolated culture expansion method of umbilical cord mesenchymal stem cells, which includes, but is not limited to, the following 4 steps:

(1) dividing the washed Wharton's jelly into 0.5-1.0mm³Centrifugally collecting the small tissues, flatly paving the small tissues on a culture substrate, and standing for 10-24 hours at the temperature of 30-40 ℃ and the humidity of 100%;

(2) slowly adding adherent culture medium 3-5 times the volume of the small tissue, wherein the adherent culture medium comprises 5-15 volume parts of serum substitute, 80-95 volume parts of alpha-MEM/DMEM-F12, 5-15ng/mL of b-FGF, 0.5-1.5% adenine and 2-3mg/mL dopamine;

Step (1)

In the present invention, step (1) is a step of leaving the tissue pieces to stand, and the inventors have found that leaving the tissue pieces to stand for a certain period of time under a humid condition without adding a culture medium or a liquid is advantageous not only for attaching the tissue pieces to a culture vessel but also for activating stem cells in the tissue to promote the stem cells to creep to the outside of the tissue. The specific reason is not clear, but may be that the stem cells are gradually in a "starved" state due to the absence of continuous supply of external energy while the tissue mass is at rest, while the tissue mass is in close contact with the culture vessel during rest to facilitate attachment of the tissue mass to the culture vessel. When the culture medium is supplemented, the stem cells in the starved state are supplemented with energy again, and the activity of the stem cells is stimulated, so that the stem cells are promoted to move out of the tissues rich in nutrition.

In the present invention, the standing time is usually 10 to 24 hours. If the standing time is too short, the degree of adherence of the tissue piece to the culture vessel tends to be weak. On the other hand, if the standing time is too long, the activity of the cells is affected due to the long-term absence of continuous energy supply, and recovery of cell viability is seriously affected, even cell death is caused. Preferred standing times may be, for example, 12 to 20 hours, such as 14, 16, 18 hours, and the like.

In the present invention, the standing condition is generally a condition suitable for the activity of the cells or tissues, and includes an appropriate temperature, and generally a temperature of 30 to 40 ℃, preferably 35 to 38 ℃, and particularly preferably 37 ℃. Considering that the tissue is in a liquid or medium free environment at rest, it is preferred to increase the humidity of the environment in which the tissue is at rest, such as a relative humidity of 90% or more, 95% or more, or even 100%.

In the present invention, a step of dividing or fragmenting the Wharton's jelly may optionally be included, with the aim of obtaining the tissue mass of the volume required by the present invention. Cutting or disrupting is generally referred to herein as physically separating the tissue of Wharton's jelly, e.g., by mechanical crushing, manual shearing, etc. To avoid disruption of the stem cells by the segmentation or disruption process, milder methods, such as manual shearing, are preferred. The process of segmentation or disruption of the Wharton's jelly preferably does not involve chemical treatment, in view of the destructive effect of chemical treatment on the stem cells. For example, no steps such as enzyme treatment are involved.

Step (1) of the present invention may further include a process of washing the divided or fragmented tissue mass or the Wharton's jelly. The washing solution used for washing is generally a physiological saline solution or a PBS buffer solution containing penicillin, streptomycin and amphotericin.

Step (1) of the present invention may further include a process of separating the divided or crushed pieces of tissue or the washed pieces of tissue, and the separation may be performed by low-speed centrifugation.

Step (2)

The step (2) of the invention is an adherence treatment step, and provides conditions for the stem cells to move out of the tissue through adherence treatment, thereby promoting the movement of the cells in the culture vessel. The adherent treatment of the present invention is carried out by using a specific adherent medium, and the amount thereof is not particularly limited and may be selected as needed, but is generally 3 to 5 times the tissue volume.

The adherent culture medium of the invention is a serum-free medium comprising a serum replacement, a basal medium and essential additional components. Wherein the essential additive components comprise b-FGF, adenine and dopamine. The basic medium is the main component, and the dosage is generally 80-95 volume parts, preferably 85-90 volume parts, such as 86 volume parts and the like. The basal medium may be alpha-MEM and/or DMEM-F12. In the present invention, the serum replacement is used in an amount of generally 5 to 15 parts by volume, preferably 6 to 12 parts by volume, more preferably 8 to 10 parts by volume. The serum replacement may be a commercially available product, preferably free of animal derived components and human platelet lysate.

In the invention, the combination of the b-FGF and the dopamine serving as necessary additional components synergistically promotes the adherent migration of cells, one of the two components has weak action on the adherent migration of the cells when the two components are used alone, and the inventor finds that the combination of the two components can greatly promote the adherent migration capability of the cells. The amount of b-FGF to be used is generally 5 to 15ng/ml, preferably 6 to 12ng/ml, more preferably 8 to 10ng/ml, in terms of concentration in the medium at the time of final use. The amount of dopamine used is typically 2-3mg/mL dopamine, based on the concentration in the medium at the end use. In the present invention, adenine as an essential additive component is used for protecting cells and promoting migration, and the amount thereof is 0.5 to 1.5%, preferably 1%, based on the concentration in the medium at the time of final use. Preferably, the adherent medium is formulated prior to use.

In the present invention, the time for the adherent treatment is generally 10 to 30 hours, preferably 15 to 25 hours, more preferably 20 to 24 hours. The adherent treatment is performed under conditions suitable for the viability of the cells or tissues, such as 37 ℃ and 5% carbon dioxide concentration.

Step (3)

Step (3) of the present invention is a primary culture step. The stem cells that have migrated in the primary culture move further in the culture vessel to form a cell layer of a flat surface. Before primary culture, the adherent culture medium is removed first, and then the primary culture medium is replaced. The primary cell culture medium is a serum-free special culture medium obtained by adding other components on the basis of a basic culture medium. Wherein, the basic culture medium can be the same as that in the adherent culture medium, or other different culture mediums in the field can be selected, and the dosage of the basic culture medium can be the same as or different from that of the basic culture medium. Other ingredients include beta-mercaptoethanol, aqueous solutions of nonessential amino acids, serum replacement, b-FGF, adenine and dopamine. One or more, preferably all, of the other ingredients described above may be used in the present invention. Wherein, the dosage and the effect of the b-FGF and the adenine can be the same as those in the adherent culture medium, but the dosage of the dopamine is required to be lower than that in the adherent culture medium. This concentration range is important for the purposes of the present invention. In primary culture, if the concentration of dopamine is too high, although the dopamine is favorable for adherence, the dopamine is not favorable for cell crawling in a culture container, and even the cell is fixed and cannot move. If the concentration is too low, the moving rate becomes low. The amount of dopamine used is generally 0.2 to 1mg/mL, based on the concentration in the medium at the time of final use, with lower concentrations, e.g., 0.2 to 0.6mg/mL, 0.3 to 0.5mg/mL, being preferred. The amount of beta-mercaptoethanol used in the present invention is generally 0.05 to 0.2 parts by volume, preferably 0.1 to 0.15 parts by volume, etc. The amount of the aqueous solution of a nonessential amino acid to be used in the present invention is generally 0.5 to 2 parts by volume, preferably 0.6 to 1.5 parts by volume, and further preferably 0.8 to 1.0 part by volume. The amount of serum replacement used in the primary culture medium is generally 5-15 parts by volume, preferably 8-12 parts by volume.

In the present invention, after the primary culture is finished, a process of collecting or harvesting the primary cells may be included, and the degree of fusion of the primary cells during collection or harvesting is generally more than 80%. Collection or pre-harvest typically involves the use of enzymes to treat the cells. In this case, the enzyme is generally a digestive enzyme, and may be a single enzyme or a complex enzyme composed of a plurality of enzymes. In the case of using the complex enzyme, the kind of the complex enzyme is not particularly limited and may be any known enzyme including, but not limited to, collagenase, hyaluronidase, dnase, trypsin.

Step (4)

In the present invention, step (4) is a subculture step for further expanding the culture to collect or harvest more desired cells. Subculture was performed by subculture medium. The subculture medium is a medium known in the art, and a known commercially available product or a medium artificially prepared according to known literature can be used.

Unless otherwise indicated, the reagents or media used in the following examples are all commercially available products.

Example 1

The composition of the medium used in this example was as follows:

adherent culture medium (prepared temporarily before use): to 90mL of DMEM-F12 were added 8mL of serum replacement and appropriate amounts of b-FGF, adenine and dopamine to concentrations of 8 ng/mL, 1% and 2mg/mL, respectively, and the pH was adjusted to 7.5.

Primary culture medium: to a medium containing 0.1mL of β -mercaptoethanol, 0.5mL of an aqueous solution of non-essential amino acids, 8mL of serum replacement, and 90mL of α -MEM/DMEM-F12 were added appropriate amounts of b-FGF, adenine and dopamine so as to give concentrations of 10ng/mL, 1% and 0.5mg/mL, respectively.

First, sample pretreatment

Taking out the aseptically obtained umbilical cord in a super clean bench, cleaning attached blood and sundries, transferring the umbilical cord to 75% alcohol for soaking and sterilizing for 1min, cleaning the sterilized umbilical cord for 3 times by using cleaning solution, then shearing off the umbilical cord along the inner sides of the ligation ends at the two sides of the umbilical cord by using tissue forceps and scissors, discarding the edematous or blood coagulation part, shearing the remaining umbilical cord into the length of 6cm +/-2 cm, extruding the blood in each section of umbilical cord by using the tissue forceps, and then transferring the umbilical cord to a new culture dish filled with the cleaning solution.

Second, obtaining and adhering wall of Wharton's jelly

Tearing the amniotic membrane on the surface of the umbilical cord along the torsion of the umbilical cord, taking out all Wharton's jelly, cleaning for 3 times by using normal saline, cutting the Wharton's jelly into small tissues with different sizes, centrifuging at low speed, collecting the small tissues, and uniformly paving the wet small tissues on the dry surface of a culture dish to ensure that the coverage of the small tissues is about 50 percent. The mixture was left to stand at 37 ℃ and 100% humidity for 24 hours without adding any medium. Next, 5ml of adherent medium was slowly added to the small pieces of tissue after standing, and incubated at 37 ℃ for 24 hours in a constant temperature incubator containing 5% carbon dioxide.

Third, primary culture

Gently removing the adherent culture medium by using a pipette, slowly adding 8ml of serum-free primary cell culture medium, continuously culturing under the same condition, replacing fresh culture medium by half a day, and observing the cell climbing-out condition after culturing for 5 days.

As a control, after small pieces of tissue were evenly plated on the surface of a dry petri dish, serum-free primary medium was slowly added directly for culture. The control culture conditions were the same as described above.

The results are shown in Table 1. As can be seen from Table 1, when the Wharton's jelly is processed to 0.5 to 1mm³The small tissue blocks can make the mesenchymal stem cells easily and quickly climb out of the tissue blocks after simultaneously standing and culturing by the adherent culture medium.

TABLE 1

Note: "/" indicates no corresponding treatment or incubation; "√" indicates that the corresponding treatment or culture is performed; the cell number was counted as the number of cells that could be visually confirmed under a microscope to be separated from the tissue mass.

Fourthly, subculturing

Observing the cell state around the tissue block in the culture bottle, performing passage amplification culture after the cell fusion degree reaches about 80%, digesting the cells by using the special mild digestive enzyme for umbilical cord mesenchymal stem cells in the passage process according to the ratio of 5000 cells/cm²Inoculating, the cell can reach 90% fusion degree after 4 days, and the extracted umbilical cord mesenchymal stem cell can stably transmit for more than 20 generations.

Example 2

And (4) carrying out flow analysis on the cell surface marker on the umbilical cord mesenchymal stem cells after the passage culture for eight generations. The results show that umbilical cord mesenchymal stem cells have better homogeneity, high expression of CD73, CD90 and CD105, and low expression of CD34, CD45, HLA-DR, CD11b and CD 19. The results are shown in FIG. 2.

Example 3

This example was used to test the effect of different components in adherent medium on the adherent effect.

First, sample pretreatment

Second, obtaining and adhering wall of Wharton's jelly

Tearing the amniotic membrane along the torsion of umbilical cord, taking out all Wharton's jelly, washing with normal saline for 3 times, and cutting into pieces of 0.5mm³Left and right small tissues are collected by low speed centrifugation and evenly spread on the surface of a dry culture dish to ensure that the coverage of the small tissues is 50% left standing at 37 ℃ and 100% humidity for 24 hours without adding any medium. Next, 5ml of adherent medium was slowly added to the small pieces of tissue after standing, and incubated at 37 ℃ for 24 hours in a constant temperature incubator containing 5% carbon dioxide. The composition of the adherent medium was obtained by adding 8ml of serum replacement and the components shown in Table 2 to 90ml of DMEM-F12.

Third, primary culture

Gently removing the adherent culture medium by using a pipette, slowly adding 8ml of serum-free primary cell culture medium, continuously culturing under the same condition, replacing a fresh adherent culture medium every day, and observing the cell climbing-out condition after culturing for 3 days. The results are shown in Table 2. The composition of the primary medium was the same as in example 1.

TABLE 2

Note: the number of cells was counted in an amount that could be visually confirmed under a microscope to be separated from the tissue mass.

Example 4

This example was used to test the effect of different components and amounts in the primary culture medium on cell climbing-out.

First, sample pretreatment

Second, obtaining and adhering wall of Wharton's jelly

Tearing the amniotic membrane along the torsion of umbilical cord, taking out all Wharton's jelly, washing with normal saline for 3 times, and cutting into pieces of 0.5mm³Left and right small pieces of tissue, low-speed centrifuging to collect small pieces of tissue, uniformly spreading on dry cultureThe dish surface was covered with a small piece of tissue at a coverage of about 50%, and left to stand at 37 ℃ and 100% humidity for 24 hours without adding any medium. Next, 5ml of adherent medium was slowly added to the small pieces of tissue after standing, and incubated at 37 ℃ for 24 hours in a constant temperature incubator containing 5% carbon dioxide. The composition of the adherent medium was the same as in example 1.

Third, primary culture

Gently removing the adherent culture medium by using a pipette, slowly adding 8ml of primary culture medium, continuously culturing under the same condition, replacing fresh primary culture medium every day, and observing the cell climbing-out condition after culturing for 3 days. The results are shown in Table 3. Composition of the primary culture medium: 0.1ml of beta-mercaptoethanol, 0.5ml of an aqueous solution of non-essential amino acids, 8ml of serum replacement, 90ml of alpha-MEM/DMEM-F12 and the components and contents shown in Table 3.

TABLE 3

Note: the number of cells was counted in an amount that could be visually confirmed under a microscope to be separated from the tissue mass. Indicates that no replacement culture of primary medium was performed for 3 days after addition of 8ml serum-free primary medium.

While the present invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. Many modifications and variations may be made to the exemplary embodiments of the present description without departing from the scope or spirit of the present invention. The scope of the claims is to be accorded the broadest interpretation so as to encompass all modifications and equivalent structures and functions.

Claims

1. A separation culture amplification method of umbilical cord mesenchymal stem cells is characterized by comprising the following steps:

(1) cutting the Wharton's jelly into 1.0mm in volume³Spreading the small tissue blocks on culture medium, and standing at 30-40 deg.C and 100% humidity for 10-24 hr;

(2) slowly adding an adherent culture medium which is 3-5 times the volume of the small tissue, wherein the adherent culture medium comprises 5-15 parts by volume of serum substitute, 80-95 parts by volume of basal culture medium, 5-15ng/mL of b-FGF, 0.5-1.5% adenine and 2-3mg/mL dopamine;

2. The isolated culture expansion method of umbilical cord mesenchymal stem cells according to claim 1, wherein the primary cell culture medium comprises 0.05-0.2 parts by volume of β -mercaptoethanol, 0.5-2 parts by volume of an aqueous solution of non-essential amino acids, 5-15 parts by volume of serum replacement, 80-95 parts by volume of α -MEM/DMEM-F12, 5-15ng/mL of b-FGF, 0.5-1.5% adenine and 0.2-1mg/mL dopamine.

3. The isolated culture expansion method of umbilical cord mesenchymal stem cells according to claim 1, wherein after collecting the small pieces of tissue, the small pieces of tissue are spread on a culture substrate without adding a culture medium or liquid.

4. The isolated culture expansion method of umbilical cord mesenchymal stem cells according to claim 1, wherein the primary cells are collected by treating the cells with digestive enzymes when the degree of fusion of the primary cells reaches 80% or more.

5. The isolated culture expansion method of umbilical cord mesenchymal stem cells according to claim 1, wherein the mesenchymal stem cells highly express CD73, CD90 and CD105, while lowly expressing or not expressing CD34, CD45, HLA-DR, CD11b and CD 19.

6. The isolated culture expansion method of umbilical cord mesenchymal stem cells according to claim 1, wherein the adherent culture medium is weakly alkaline at least at the time of initial contact with the tissue mass.

7. The isolated culture expansion method of umbilical cord mesenchymal stem cells according to claim 6, wherein the weak alkaline means that the pH is in the range of 7.5-7.8.

8. The isolated culture expansion method of umbilical cord mesenchymal stem cells according to claim 1, wherein the Wharton's jelly is washed using a washing solution, the washing solution being a physiological saline solution or PBS buffer solution containing penicillin, streptomycin and amphotericin.

9. The isolated culture expansion method of umbilical cord mesenchymal stem cells according to claim 1, wherein the adherent culture medium is free of animal derived components and human platelet lysate.

10. An umbilical cord mesenchymal stem cell characterized by being prepared by the amplification method according to any one of claims 1 to 9.