CN113249313A - Primary culture method of stem cells with high cell adherence capacity - Google Patents

Primary culture method of stem cells with high cell adherence capacity Download PDF

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CN113249313A
CN113249313A CN202110530444.6A CN202110530444A CN113249313A CN 113249313 A CN113249313 A CN 113249313A CN 202110530444 A CN202110530444 A CN 202110530444A CN 113249313 A CN113249313 A CN 113249313A
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tissue
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primary culture
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朱灏
葛晨曦
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Dassel Shanghai Life Technology Co ltd
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Dassel Shanghai Life Technology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0668—Mesenchymal stem cells from other natural sources
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • C12N2509/10—Mechanical dissociation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/52—Fibronectin; Laminin

Abstract

The invention discloses a primary culture method of stem cells with high cell adherence capacity, which uses a primary culture medium with definite components and does not contain serum, avoids the uncertainty of the components of the serum culture medium and the instability of serum culture, simultaneously improves the cell adherence capacity in the serum-free culture medium, reduces the adherence time, improves the efficiency, and the cells of a working library prepared from the human umbilical cord mesenchymal stem cells in the primary culture have good morphology.

Description

Primary culture method of stem cells with high cell adherence capacity
Technical Field
The invention belongs to the technical field of cell biology, and particularly relates to a primary culture method of stem cells with high cell adherence capacity.
Background
Umbilical cord mesenchymal Stem Cells (UC-MSCs) refer to a pluripotent Stem cell isolated from Umbilical cord tissue of a newborn. Can be differentiated to form various mesoderm cells such as bone, cartilage, muscle, fat and the like. Compared with the bone marrow stem cells, the UC-MSCs have rich sources and low immunogenicity, and are relatively ideal sources of seed cells.
As a common cell in regenerative medicine, in vitro culture and amplification of UC-MSCs are necessary conditions for promoting the clinical research make internal disorder or usurp and application thereof. Factors influencing the culture and amplification of the UC-MSCs comprise a culture method and a culture medium, and in the in-vitro culture process, the serum-free culture medium with clear components can avoid the uncertainty of the components of the serum culture medium and the instability of the serum culture, thereby being beneficial to researchers to accurately regulating and controlling the cell behavior. However, the cell adherence ability in the serum-free culture medium is weak, so that the primary culture of the umbilical cord mesenchymal stem cells is influenced, and the subsequent subculture is influenced.
Disclosure of Invention
In order to solve the above problems, the present invention provides a primary culture method of stem cells with high cell adherence capacity, which at least comprises the following steps:
s1, clipping the umbilical cord, transferring the umbilical cord into a culture dish, extruding residual blood and clipping; then transferring the tissue to a new culture dish to strip off the blood vessel and the adventitia to obtain a Wharton jelly tissue;
s2, placing the Wharton jelly tissue in a new culture dish, and adding sodium chloride injection for cleaning; transferring into a centrifuge tube, and cutting into small pieces; sucking Whitmania lacustra tissue paste and evenly spreading the Whitmania lacustra tissue paste at the bottom of the culture bottle;
s3, placing the culture bottle in a biological safety cabinet for standing, adding a primary culture medium into the culture bottle after the tissue adheres to the wall, and slowly shaking the bottle body to enable the culture medium to completely cover the bottom surface; placing the culture bottle into an incubator for culture.
As a preferable technical scheme, the sodium chloride injection in S2 is washed for 8-10 times.
As an optimal technical scheme, the volume of the small blocks cut in S2 is 1-2 mm3。
As a preferable technical scheme, the culture condition of the culture box is 37.0 ℃ and 5.0% CO2。
As a preferred technical scheme, the primary culture medium at least comprises the following components: serum-free medium of mesenchymal stem cells, fibronectin.
As a preferable technical scheme, the primary culture medium at least comprises the following components in parts by weight: 98-100 parts of mesenchymal stem cell serum-free medium and 0-0.5 part of fibronectin.
As a preferable technical scheme, the primary culture medium at least comprises the following components in parts by weight: 100 parts of mesenchymal stem cell serum-free medium and 0.25 part of fibronectin.
As a preferred embodiment, the culture method further comprises: and (3) putting the culture bottle into an incubator for culturing for 4-6 days, supplementing a primary culture medium into the culture bottle, and continuing culturing.
As a preferred embodiment, the culture method further comprises: after the culture bottle is placed into an incubator to be cultured for 7-9 days, a pipette is used for sucking and removing the supernatant fluid and the fallen tissue blocks of the culture bottle, and then primary culture medium is added for continuous culture; the amount of the primary medium added thereto was 2 times as much as the amount of the primary medium added in S1.
The invention also provides a primary cultured human umbilical cord mesenchymal stem cell which is obtained by the primary culture method of the stem cell with high adherence capability.
Has the advantages that:
the invention provides a primary culture method of stem cells with high cell adherence capacity, which uses a primary culture medium with definite components and does not contain serum, avoids the uncertainty of the components of the serum culture medium and the instability of serum culture, simultaneously improves the cell adherence capacity in the serum-free culture medium, reduces the adherence time, improves the efficiency, and the cells of a working library prepared from the human umbilical cord mesenchymal stem cells in the primary culture have good morphology.
Drawings
FIG. 1 is a morphological diagram of cells of a working bank prepared from primary cultured human umbilical cord mesenchymal stem cells of example 1.
Detailed Description
The invention will be further understood by reference to the following detailed description of preferred embodiments of the invention and the examples included therein. 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. To the extent that a definition of a particular term disclosed in the prior art is inconsistent with any definition provided in the present disclosure, the definition of the term provided in the present disclosure controls.
As used herein, a feature that does not define a singular or plural form is also intended to include a plural form of the feature unless the context clearly indicates otherwise. It will be further understood that the term "prepared from …," as used herein, is synonymous with "comprising," including, "comprising," "having," "including," and/or "containing," when used in this specification means that the recited composition, step, method, article, or device is present, but does not preclude the presence or addition of one or more other compositions, steps, methods, articles, or devices. Furthermore, the use of "preferred," "preferably," "more preferred," etc., when describing embodiments of the present invention, is meant to refer to embodiments of the invention that may provide certain benefits, under certain circumstances. However, other embodiments may be preferred, under the same or other circumstances. In addition, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.
In order to solve the above problems, a first aspect of the present invention provides a method for primary culture of stem cells with high cell adherence capacity, comprising at least the following steps:
s1, clipping the umbilical cord, transferring the umbilical cord into a culture dish, extruding residual blood and clipping; then transferring the tissue to a new culture dish to strip off the blood vessel and the adventitia to obtain a Wharton jelly tissue;
s2, placing the Wharton jelly tissue in a new culture dish, and adding sodium chloride injection for cleaning; transferring into a centrifuge tube, and cutting into small pieces; sucking Whitmania lacustra tissue paste and evenly spreading the Whitmania lacustra tissue paste at the bottom of the culture bottle;
s3, placing the culture bottle in a biological safety cabinet for standing, adding a primary culture medium into the culture bottle after the tissue adheres to the wall, and slowly shaking the bottle body to enable the culture medium to completely cover the bottom surface; placing the culture bottle into an incubator for culture.
In some preferred embodiments, the sodium chloride injection is washed 8-10 times in S2.
In some preferred embodiments, the volume of the small pieces cut in S2 is 1-2 mm3。
In some preferred embodiments, the culture conditions of the incubator are 37.0 ℃ and 5.0% CO2。
The clinical requirements for the human umbilical cord mesenchymal stem cells for treatment are as follows: the cell preparation has no risk of infection of pathogenic factors; serum-free culture medium with definite additive components is required for cell amplification, residual culture medium has no adverse effect on receptors, and antibiotics are not used in the culture process; the cell preparation should contain sufficient cells to maintain high viability and effectively maintain the biological properties associated with the treatment. According to these criteria, the conventional serum-containing medium for amplifying human umbilical cord mesenchymal stem cells has failed to satisfy clinical requirements. Although some biological companies have developed several commercial serum-free media suitable for in vitro amplification, there still exist problems in use, such as weak cell anchorage ability during primary culture, and reduced cell surface-specific antigen expression, differentiation potential, etc. after culture.
In order to increase the cell anchorage and antioxidant capacity during primary culture, in some preferred embodiments, the primary culture medium comprises at least the following components: serum-free medium of mesenchymal stem cells, fibronectin.
In some preferred embodiments, the primary culture medium comprises, in parts by weight, at least the following components: 98-100 parts of mesenchymal stem cell serum-free medium and 0-0.5 part of fibronectin.
In some preferred embodiments, the primary culture medium comprises, in parts by weight, at least the following components: 100 parts of mesenchymal stem cell serum-free medium and 0.25 part of fibronectin.
Fibronectin passes through adhesion sites on fibronectin molecules, affecting cell adhesion, migration, etc., thereby increasing cell anchorage. When 98-100 parts of the serum-free culture medium of the mesenchymal stem cells and 0-0.5 part of fibronectin are used, the inventor unexpectedly finds that the cell adherence capacity is further enhanced, and the morphological structure of the cells is better, which probably results from the combination of the cells and adhesion sites on fibronectin molecules, shortens the cell confluence time, and improves the stability of an extracellular matrix, thereby improving the cell adherence rate, the confluence rate and the structural stability of the cells.
In some preferred embodiments, the culturing method further comprises: and (3) putting the culture bottle into an incubator for culturing for 4-6 days, supplementing a primary culture medium into the culture bottle, and continuing culturing.
In some preferred embodiments, the culturing method further comprises: after the culture bottle is placed into an incubator to be cultured for 7-9 days, a pipette is used for sucking and removing the supernatant fluid and the fallen tissue blocks of the culture bottle, and then primary culture medium is added for continuous culture; the amount of the primary medium added thereto was 2 times as much as the amount of the primary medium added in S1.
The invention also provides a primary cultured human umbilical cord mesenchymal stem cell which is obtained by the primary culture method of the stem cell with high adherence capability.
The present invention will be specifically described below by way of examples. It should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and that the insubstantial modifications and adaptations of the present invention by those skilled in the art based on the above disclosure are still within the scope of the present invention.
In addition, the starting materials used are all commercially available, unless otherwise specified.
Examples
The technical solution of the present invention is described in detail by the following examples, but the scope of the present invention is not limited to the examples.
Example 1
Embodiment 1 provides a method for primary culture of stem cells with high cell adherence capacity, comprising the following steps:
s1, taking the umbilical cord out of the collection bottle, putting the umbilical cord into a 150mm culture dish filled with 30ml of sodium chloride injection, and shearing umbilical cord tissues about 10 cm. Transferring the cut umbilical cord tissues into a new 150mm culture dish filled with 30ml of sodium chloride injection, and extruding and cutting residual blood in the umbilical cord by using hemostatic forceps. The umbilical cord segments are transferred into a new 150mm culture dish filled with 30ml of sodium chloride injection, and umbilical cord blood vessels and adventitia are peeled off to obtain the Wharton's jelly tissues.
S2, placing the collected Wharton' S jelly tissues in a new 150mm culture dish, and adding a proper amount of sodium chloride injection to wash for 8 times. Transferring the washed Wharton jelly tissue into a 50ml centrifuge tube, and shearing the Wharton jelly tissue into a tissue with the volume of 1-2 mm by using ophthalmic scissors3Small pieces of (a). Sucking 0.5ml of Whistle's jelly tissue paste with a Pasteur pipetteSlurrying at 75cm2In the flask, the tissue was evenly spread with a 5ml pipette on the bottom of the flask.
And S3, placing the culture bottle in a biological safety cabinet for standing until all tissues adhere to the wall. Marking of adherence: the culture bottle is reversely buckled, the tissue cannot slip, and the tissue is in contact with the bottom of the bottle without water stain visible to naked eyes.
After the tissue adheres to the wall, 5ml of primary culture medium is added into the culture bottle, and the bottle body is slowly shaken after the primary culture medium is added, so that the culture medium completely covers the bottom surface. Placing the culture flask at 37.0 deg.C and 5.0% CO2Culturing in an incubator.
S4, on day 5 after the tissue piece was inoculated, the cultured cells were taken out from the incubator. And (4) observing the growth condition of the cells under a microscope, and if spindle-shaped adherent cells appear around the tissue block, performing fluid infusion operation, and supplementing 5ml of primary culture medium into the culture flask. After the operation was completed, the flask was placed at 37.0 ℃ with 5.0% CO2And continuing culturing in the incubator.
S5, on day 8 after the tissue block was inoculated, the cultured cells were taken out from the incubator. The growth of the cells was observed under a microscope, and if it was found that more than 30% of the tissue pieces had cells on the edges, the medium was changed, and after the culture supernatant and the detached tissue pieces were removed by pipette, 10ml of the primary medium was added to the culture flask. After the operation was completed, the flask was placed at 37.0 ℃ with 5.0% CO2Continuously culturing in an incubator; when more than 50% of the tissue blocks are observed to climb out of the cells around the tissue blocks under a microscope and the local fusion degree is more than 80%, the primary culture is completed.
The primary culture medium at least comprises the following components in parts by weight: 100 parts of mesenchymal stem cell serum-free medium and 0.25 part of fibronectin.
The mesenchymal stem cell serum-free culture medium is purchased from Wuhan Punuo Sai Life technologies, Inc., with the product number of CM-SC 01;
the fibronectin was purchased from Shanghai leaf Biotech, Inc. under the trade designation S12073-5 mg.
Embodiment 1 also provides a primary cultured human umbilical cord mesenchymal stem cell, which is obtained by culturing the above-mentioned primary culture method of stem cells with high anchorage ability.
Example 2
Embodiment 2 provides a method for primary culture of stem cells with high cell adherence capacity, comprising the following steps:
s1, taking the umbilical cord out of the collection bottle, putting the umbilical cord into a 150mm culture dish filled with 30ml of sodium chloride injection, and shearing umbilical cord tissues about 10 cm. Transferring the cut umbilical cord tissues into a new 150mm culture dish filled with 30ml of sodium chloride injection, and extruding and cutting residual blood in the umbilical cord by using hemostatic forceps. The umbilical cord segments are transferred into a new 150mm culture dish filled with 30ml of sodium chloride injection, and umbilical cord blood vessels and adventitia are peeled off to obtain the Wharton's jelly tissues.
S2, placing the collected Wharton' S jelly tissues in a new 150mm culture dish, and adding a proper amount of sodium chloride injection to wash for 8 times. Transferring the washed Wharton jelly tissue into a 50ml centrifuge tube, and shearing the Wharton jelly tissue into a tissue with the volume of 1-2 mm by using ophthalmic scissors3Small pieces of (a). Sucking 0.5ml of Whistle's jelly tissue paste to 75cm with a Pasteur pipette2In the flask, the tissue was evenly spread with a 5ml pipette on the bottom of the flask.
And S3, placing the culture bottle in a biological safety cabinet for standing until all tissues adhere to the wall. Marking of adherence: the culture bottle is reversely buckled, the tissue cannot slip, and the tissue is in contact with the bottom of the bottle without water stain visible to naked eyes.
After the tissue adheres to the wall, 5ml of primary culture medium is added into the culture bottle, and the bottle body is slowly shaken after the primary culture medium is added, so that the culture medium completely covers the bottom surface. Placing the culture flask at 37.0 deg.C and 5.0% CO2Culturing in an incubator.
S4, on day 5 after the tissue piece was inoculated, the cultured cells were taken out from the incubator. And (4) observing the growth condition of the cells under a microscope, and if spindle-shaped adherent cells appear around the tissue block, performing fluid infusion operation, and supplementing 5ml of primary culture medium into the culture flask. After the operation was completed, the flask was placed at 37.0 ℃ with 5.0% CO2And continuing culturing in the incubator.
S5, on the 8 th day after the tissue piece inoculation, the cultured fine particles were taken out from the incubatorAnd (4) cells. The growth of the cells was observed under a microscope, and if it was found that more than 30% of the tissue pieces had cells on the edges, the medium was changed, and after the culture supernatant and the detached tissue pieces were removed by pipette, 10ml of the primary medium was added to the culture flask. After the operation was completed, the flask was placed at 37.0 ℃ with 5.0% CO2Continuously culturing in an incubator; when more than 50% of the tissue blocks are observed to climb out of the cells around the tissue blocks under a microscope and the local fusion degree is more than 80%, the primary culture is completed.
The primary culture medium at least comprises the following components in parts by weight: 98 parts of mesenchymal stem cell serum-free medium and 0.25 part of fibronectin.
The mesenchymal stem cell serum-free culture medium is purchased from Wuhan Punuo Sai Life technologies, Inc., with the product number of CM-SC 01;
the fibronectin was purchased from Shanghai leaf Biotech, Inc. under the trade designation S12073-5 mg.
Embodiment 2 also provides a primary cultured human umbilical cord mesenchymal stem cell, which is obtained by culturing the above-mentioned primary culture method of stem cells with high anchorage ability.
Example 3
Embodiment 3 provides a method for primary culture of stem cells with high cell adherence capacity, comprising the following steps:
s1, taking the umbilical cord out of the collection bottle, putting the umbilical cord into a 150mm culture dish filled with 30ml of sodium chloride injection, and shearing umbilical cord tissues about 10 cm. Transferring the cut umbilical cord tissues into a new 150mm culture dish filled with 30ml of sodium chloride injection, and extruding and cutting residual blood in the umbilical cord by using hemostatic forceps. The umbilical cord segments are transferred into a new 150mm culture dish filled with 30ml of sodium chloride injection, and umbilical cord blood vessels and adventitia are peeled off to obtain the Wharton's jelly tissues.
S2, placing the collected Wharton' S jelly tissues in a new 150mm culture dish, and adding a proper amount of sodium chloride injection to wash for 8 times. Transferring the washed Wharton jelly tissue into a 50ml centrifuge tube, and shearing the Wharton jelly tissue into a tissue with the volume of 1-2 mm by using ophthalmic scissors3Small pieces of (a). Sucking 0.5ml of Whistle's jelly tissue paste to 75cm with a Pasteur pipette2CulturingIn the flask, the tissue was evenly spread on the bottom of the flask with a 5ml pipette.
And S3, placing the culture bottle in a biological safety cabinet for standing until all tissues adhere to the wall. Marking of adherence: the culture bottle is reversely buckled, the tissue cannot slip, and the tissue is in contact with the bottom of the bottle without water stain visible to naked eyes.
After the tissue adheres to the wall, 5ml of primary culture medium is added into the culture bottle, and the bottle body is slowly shaken after the primary culture medium is added, so that the culture medium completely covers the bottom surface. Placing the culture flask at 37.0 deg.C and 5.0% CO2Culturing in an incubator.
S4, on day 5 after the tissue piece was inoculated, the cultured cells were taken out from the incubator. And (4) observing the growth condition of the cells under a microscope, and if spindle-shaped adherent cells appear around the tissue block, performing fluid infusion operation, and supplementing 5ml of primary culture medium into the culture flask. After the operation was completed, the flask was placed at 37.0 ℃ with 5.0% CO2And continuing culturing in the incubator.
S5, on day 8 after the tissue block was inoculated, the cultured cells were taken out from the incubator. The growth of the cells was observed under a microscope, and if it was found that more than 30% of the tissue pieces had cells on the edges, the medium was changed, and after the culture supernatant and the detached tissue pieces were removed by pipette, 10ml of the primary medium was added to the culture flask. After the operation was completed, the flask was placed at 37.0 ℃ with 5.0% CO2Continuously culturing in an incubator; when more than 50% of the tissue blocks are observed to climb out of the cells around the tissue blocks under a microscope and the local fusion degree is more than 80%, the primary culture is completed.
The primary culture medium at least comprises the following components in parts by weight: 99 parts of mesenchymal stem cell serum-free medium and 0.25 part of fibronectin.
The mesenchymal stem cell serum-free culture medium is purchased from Wuhan Punuo Sai Life technologies, Inc., with the product number of CM-SC 01;
the fibronectin was purchased from Shanghai leaf Biotech, Inc. under the trade designation S12073-5 mg.
Embodiment 3 also provides a primary cultured human umbilical cord mesenchymal stem cell, which is obtained by culturing the above-mentioned primary culture method of stem cells with high anchorage ability.
Example 4
Embodiment 4 provides a method for primary culture of stem cells with high cell adherence capacity, comprising the following steps:
s1, taking the umbilical cord out of the collection bottle, putting the umbilical cord into a 150mm culture dish filled with 30ml of sodium chloride injection, and shearing umbilical cord tissues about 10 cm. Transferring the cut umbilical cord tissues into a new 150mm culture dish filled with 30ml of sodium chloride injection, and extruding and cutting residual blood in the umbilical cord by using hemostatic forceps. The umbilical cord segments are transferred into a new 150mm culture dish filled with 30ml of sodium chloride injection, and umbilical cord blood vessels and adventitia are peeled off to obtain the Wharton's jelly tissues.
S2, placing the collected Wharton' S jelly tissues in a new 150mm culture dish, and adding a proper amount of sodium chloride injection to wash for 8 times. Transferring the washed Wharton jelly tissue into a 50ml centrifuge tube, and shearing the Wharton jelly tissue into a tissue with the volume of 1-2 mm by using ophthalmic scissors3Small pieces of (a). Sucking 0.5ml of Whistle's jelly tissue paste to 75cm with a Pasteur pipette2In the flask, the tissue was evenly spread with a 5ml pipette on the bottom of the flask.
And S3, placing the culture bottle in a biological safety cabinet for standing until all tissues adhere to the wall. Marking of adherence: the culture bottle is reversely buckled, the tissue cannot slip, and the tissue is in contact with the bottom of the bottle without water stain visible to naked eyes.
After the tissue adheres to the wall, 5ml of primary culture medium is added into the culture bottle, and the bottle body is slowly shaken after the primary culture medium is added, so that the culture medium completely covers the bottom surface. Placing the culture flask at 37.0 deg.C and 5.0% CO2Culturing in an incubator.
S4, on day 5 after the tissue piece was inoculated, the cultured cells were taken out from the incubator. And (4) observing the growth condition of the cells under a microscope, and if spindle-shaped adherent cells appear around the tissue block, performing fluid infusion operation, and supplementing 5ml of primary culture medium into the culture flask. After the operation was completed, the flask was placed at 37.0 ℃ with 5.0% CO2And continuing culturing in the incubator.
S5, on day 8 after the tissue block was inoculated, the cultured cells were taken out from the incubator. Observation of cells under microscopeIn the case of growth, when cell climbing out from the edge of more than 30% of the tissue piece was observed, the medium was changed, and after the culture supernatant and the detached tissue piece were aspirated by a pipette, 10ml of the primary medium was added to the culture flask. After the operation was completed, the flask was placed at 37.0 ℃ with 5.0% CO2Continuously culturing in an incubator; when more than 50% of the tissue blocks are observed to climb out of the cells around the tissue blocks under a microscope and the local fusion degree is more than 80%, the primary culture is completed.
The primary culture medium at least comprises the following components in parts by weight: 100 parts of mesenchymal stem cell serum-free medium and 0.2 part of fibronectin.
The mesenchymal stem cell serum-free culture medium is purchased from Wuhan Punuo Sai Life technologies, Inc., with the product number of CM-SC 01;
the fibronectin was purchased from Shanghai leaf Biotech, Inc. under the trade designation S12073-5 mg.
Embodiment 4 also provides a primary cultured human umbilical cord mesenchymal stem cell, which is obtained by culturing the above-mentioned primary culture method of stem cells with high anchorage ability.
Example 5
Embodiment 5 provides a method for primary culture of stem cells with high cell adherence capacity, comprising the following steps:
s1, taking the umbilical cord out of the collection bottle, putting the umbilical cord into a 150mm culture dish filled with 30ml of sodium chloride injection, and shearing umbilical cord tissues about 10 cm. Transferring the cut umbilical cord tissues into a new 150mm culture dish filled with 30ml of sodium chloride injection, and extruding and cutting residual blood in the umbilical cord by using hemostatic forceps. The umbilical cord segments are transferred into a new 150mm culture dish filled with 30ml of sodium chloride injection, and umbilical cord blood vessels and adventitia are peeled off to obtain the Wharton's jelly tissues.
S2, placing the collected Wharton' S jelly tissues in a new 150mm culture dish, and adding a proper amount of sodium chloride injection to wash for 8 times. Transferring the washed Wharton jelly tissue into a 50ml centrifuge tube, and shearing the Wharton jelly tissue into a tissue with the volume of 1-2 mm by using ophthalmic scissors3Small pieces of (a). Sucking 0.5ml of Whistle's jelly tissue paste to 75cm with a Pasteur pipette2In a culture flask, the group was pipetted with a 5ml pipetteEvenly weaving and flatly paving the tissue at the bottom of the culture bottle.
And S3, placing the culture bottle in a biological safety cabinet for standing until all tissues adhere to the wall. Marking of adherence: the culture bottle is reversely buckled, the tissue cannot slip, and the tissue is in contact with the bottom of the bottle without water stain visible to naked eyes.
After the tissue adheres to the wall, 5ml of primary culture medium is added into the culture bottle, and the bottle body is slowly shaken after the primary culture medium is added, so that the culture medium completely covers the bottom surface. Placing the culture flask at 37.0 deg.C and 5.0% CO2Culturing in an incubator.
S4, on day 5 after the tissue piece was inoculated, the cultured cells were taken out from the incubator. And (4) observing the growth condition of the cells under a microscope, and if spindle-shaped adherent cells appear around the tissue block, performing fluid infusion operation, and supplementing 5ml of primary culture medium into the culture flask. After the operation was completed, the flask was placed at 37.0 ℃ with 5.0% CO2And continuing culturing in the incubator.
S5, on day 8 after the tissue block was inoculated, the cultured cells were taken out from the incubator. The growth of the cells was observed under a microscope, and if it was found that more than 30% of the tissue pieces had cells on the edges, the medium was changed, and after the culture supernatant and the detached tissue pieces were removed by pipette, 10ml of the primary medium was added to the culture flask. After the operation was completed, the flask was placed at 37.0 ℃ with 5.0% CO2Continuously culturing in an incubator; when more than 50% of the tissue blocks are observed to climb out of the cells around the tissue blocks under a microscope and the local fusion degree is more than 80%, the primary culture is completed.
The primary culture medium at least comprises the following components in parts by weight: 100 parts of mesenchymal stem cell serum-free medium and 0.15 part of fibronectin.
The mesenchymal stem cell serum-free culture medium is purchased from Wuhan Punuo Sai Life technologies, Inc., with the product number of CM-SC 01;
the fibronectin was purchased from Shanghai leaf Biotech, Inc. under the trade designation S12073-5 mg.
Embodiment 5 also provides a primary cultured human umbilical cord mesenchymal stem cell, which is obtained by culturing the above-mentioned primary culture method of stem cells with high anchorage ability.
Comparative example 1
Comparative example 1 provides a primary culture method of stem cells with high cell adherence capacity, comprising the following steps:
s1, taking the umbilical cord out of the collection bottle, putting the umbilical cord into a 150mm culture dish filled with 30ml of sodium chloride injection, and shearing umbilical cord tissues about 10 cm. Transferring the cut umbilical cord tissues into a new 150mm culture dish filled with 30ml of sodium chloride injection, and extruding and cutting residual blood in the umbilical cord by using hemostatic forceps. The umbilical cord segments are transferred into a new 150mm culture dish filled with 30ml of sodium chloride injection, and umbilical cord blood vessels and adventitia are peeled off to obtain the Wharton's jelly tissues.
S2, placing the collected Wharton' S jelly tissues in a new 150mm culture dish, and adding a proper amount of sodium chloride injection to wash for 8 times. Transferring the washed Wharton jelly tissue into a 50ml centrifuge tube, and shearing the Wharton jelly tissue into a tissue with the volume of 1-2 mm by using ophthalmic scissors3Small pieces of (a). Sucking 0.5ml of Whistle's jelly tissue paste to 75cm with a Pasteur pipette2In the flask, the tissue was evenly spread with a 5ml pipette on the bottom of the flask.
And S3, placing the culture bottle in a biological safety cabinet for standing until all tissues adhere to the wall. Marking of adherence: the culture bottle is reversely buckled, the tissue cannot slip, and the tissue is in contact with the bottom of the bottle without water stain visible to naked eyes.
After the tissue adheres to the wall, 5ml of primary culture medium is added into the culture bottle, and the bottle body is slowly shaken after the primary culture medium is added, so that the culture medium completely covers the bottom surface. Placing the culture flask at 37.0 deg.C and 5.0% CO2Culturing in an incubator.
S4, on day 5 after the tissue piece was inoculated, the cultured cells were taken out from the incubator. And (4) observing the growth condition of the cells under a microscope, and if spindle-shaped adherent cells appear around the tissue block, performing fluid infusion operation, and supplementing 5ml of primary culture medium into the culture flask. After the operation was completed, the flask was placed at 37.0 ℃ with 5.0% CO2And continuing culturing in the incubator.
S5, on day 8 after the tissue block was inoculated, the cultured cells were taken out from the incubator. Observing the growth of the cells under a microscope, and if the growth is found to be 30 percentWhen there were cells on the edge of the above tissue piece, the liquid was changed, and after the culture supernatant and the detached tissue piece were aspirated by a pipette, 10ml of primary culture medium was added to the culture flask. After the operation was completed, the flask was placed at 37.0 ℃ with 5.0% CO2Continuously culturing in an incubator; when more than 50% of the tissue blocks are observed to climb out of the cells around the tissue blocks under a microscope and the local fusion degree is more than 80%, the primary culture is completed.
The primary culture medium at least comprises the following components in parts by weight: 100 parts of mesenchymal stem cell serum-free culture medium.
The mesenchymal stem cell serum-free medium is purchased from Wuhan Punuo Sai Life technologies, Inc., and has a product number of CM-SC 01.
The comparative example 1 also provides a primary cultured human umbilical cord mesenchymal stem cell which is obtained by the primary culture method of the stem cell with high cell adherence capability.
Comparative example 2
Comparative example 2 provides a primary culture method of stem cells with high cell adherence capacity, comprising the following steps:
s1, taking the umbilical cord out of the collection bottle, putting the umbilical cord into a 150mm culture dish filled with 30ml of sodium chloride injection, and shearing umbilical cord tissues about 10 cm. Transferring the cut umbilical cord tissues into a new 150mm culture dish filled with 30ml of sodium chloride injection, and extruding and cutting residual blood in the umbilical cord by using hemostatic forceps. The umbilical cord segments are transferred into a new 150mm culture dish filled with 30ml of sodium chloride injection, and umbilical cord blood vessels and adventitia are peeled off to obtain the Wharton's jelly tissues.
S2, placing the collected Wharton' S jelly tissues in a new 150mm culture dish, and adding a proper amount of sodium chloride injection to wash for 8 times. Transferring the washed Wharton jelly tissue into a 50ml centrifuge tube, and shearing the Wharton jelly tissue into a tissue with the volume of 1-2 mm by using ophthalmic scissors3Small pieces of (a). Sucking 0.5ml of Whistle's jelly tissue paste to 75cm with a Pasteur pipette2In the flask, the tissue was evenly spread with a 5ml pipette on the bottom of the flask.
And S3, placing the culture bottle in a biological safety cabinet for standing until all tissues adhere to the wall. Marking of adherence: the culture bottle is reversely buckled, the tissue cannot slip, and the tissue is in contact with the bottom of the bottle without water stain visible to naked eyes.
After the tissue adheres to the wall, 5ml of primary culture medium is added into the culture bottle, and the bottle body is slowly shaken after the primary culture medium is added, so that the culture medium completely covers the bottom surface. Placing the culture flask at 37.0 deg.C and 5.0% CO2Culturing in an incubator.
S4, on day 5 after the tissue piece was inoculated, the cultured cells were taken out from the incubator. And (4) observing the growth condition of the cells under a microscope, and if spindle-shaped adherent cells appear around the tissue block, performing fluid infusion operation, and supplementing 5ml of primary culture medium into the culture flask. After the operation was completed, the flask was placed at 37.0 ℃ with 5.0% CO2And continuing culturing in the incubator.
S5, on day 8 after the tissue block was inoculated, the cultured cells were taken out from the incubator. The growth of the cells was observed under a microscope, and if it was found that more than 30% of the tissue pieces had cells on the edges, the medium was changed, and after the culture supernatant and the detached tissue pieces were removed by pipette, 10ml of the primary medium was added to the culture flask. After the operation was completed, the flask was placed at 37.0 ℃ with 5.0% CO2Continuously culturing in an incubator; when more than 50% of the tissue blocks are observed to climb out of the cells around the tissue blocks under a microscope and the local fusion degree is more than 80%, the primary culture is completed.
The primary culture medium at least comprises the following components in parts by weight: 90 parts of mesenchymal stem cell serum-free medium and 0.25 part of fibronectin.
The mesenchymal stem cell serum-free culture medium is purchased from Wuhan Punuo Sai Life technologies, Inc., with the product number of CM-SC 01;
the comparative example 2 also provides a primary cultured human umbilical cord mesenchymal stem cell which is obtained by the primary culture method of the stem cell with high cell adherence capability.
Comparative example 3
Comparative example 3 provides a primary culture method of stem cells with high cell adherence capacity, comprising the following steps:
s1, taking the umbilical cord out of the collection bottle, putting the umbilical cord into a 150mm culture dish filled with 30ml of sodium chloride injection, and shearing umbilical cord tissues about 10 cm. Transferring the cut umbilical cord tissues into a new 150mm culture dish filled with 30ml of sodium chloride injection, and extruding and cutting residual blood in the umbilical cord by using hemostatic forceps. The umbilical cord segments are transferred into a new 150mm culture dish filled with 30ml of sodium chloride injection, and umbilical cord blood vessels and adventitia are peeled off to obtain the Wharton's jelly tissues.
S2, placing the collected Wharton' S jelly tissues in a new 150mm culture dish, and adding a proper amount of sodium chloride injection to wash for 8 times. Transferring the washed Wharton jelly tissue into a 50ml centrifuge tube, and shearing the Wharton jelly tissue into a tissue with the volume of 1-2 mm by using ophthalmic scissors3Small pieces of (a). Sucking 0.5ml of Whistle's jelly tissue paste to 75cm with a Pasteur pipette2In the flask, the tissue was evenly spread with a 5ml pipette on the bottom of the flask.
And S3, placing the culture bottle in a biological safety cabinet for standing until all tissues adhere to the wall. Marking of adherence: the culture bottle is reversely buckled, the tissue cannot slip, and the tissue is in contact with the bottom of the bottle without water stain visible to naked eyes.
After the tissue adheres to the wall, 5ml of primary culture medium is added into the culture bottle, and the bottle body is slowly shaken after the primary culture medium is added, so that the culture medium completely covers the bottom surface. Placing the culture flask at 37.0 deg.C and 5.0% CO2Culturing in an incubator.
S4, on day 5 after the tissue piece was inoculated, the cultured cells were taken out from the incubator. And (4) observing the growth condition of the cells under a microscope, and if spindle-shaped adherent cells appear around the tissue block, performing fluid infusion operation, and supplementing 5ml of primary culture medium into the culture flask. After the operation was completed, the flask was placed at 37.0 ℃ with 5.0% CO2And continuing culturing in the incubator.
S5, on day 8 after the tissue block was inoculated, the cultured cells were taken out from the incubator. The growth of the cells was observed under a microscope, and if it was found that more than 30% of the tissue pieces had cells on the edges, the medium was changed, and after the culture supernatant and the detached tissue pieces were removed by pipette, 10ml of the primary medium was added to the culture flask. After the operation was completed, the flask was placed at 37.0 ℃ with 5.0% CO2Continuously culturing in an incubator; when observed under a microscope, the content of the active carbon is more than 50 percentThe cells are crawled out from the periphery of the tissue block, and the local fusion degree is more than 80 percent, so that the primary culture is completed.
The primary culture medium at least comprises the following components in parts by weight: 100 parts of mesenchymal stem cell serum-free medium and 0.8 part of fibronectin.
The mesenchymal stem cell serum-free culture medium is purchased from Wuhan Punuo Sai Life technologies, Inc., with the product number of CM-SC 01;
the fibronectin was purchased from Shanghai leaf Biotech, Inc. under the trade designation S12073-5 mg.
The comparative example 3 also provides a primary cultured human umbilical cord mesenchymal stem cell which is obtained by the primary culture method of the stem cell with high cell adherence capability.
Comparative example 4
Comparative example 4 provides a primary culture method of stem cells with high cell adherence capacity, comprising the following steps:
s1, taking the umbilical cord out of the collection bottle, putting the umbilical cord into a 150mm culture dish filled with 30ml of sodium chloride injection, and shearing umbilical cord tissues about 10 cm. Transferring the cut umbilical cord tissues into a new 150mm culture dish filled with 30ml of sodium chloride injection, and extruding and cutting residual blood in the umbilical cord by using hemostatic forceps. The umbilical cord segments are transferred into a new 150mm culture dish filled with 30ml of sodium chloride injection, and umbilical cord blood vessels and adventitia are peeled off to obtain the Wharton's jelly tissues.
S2, placing the collected Wharton' S jelly tissues in a new 150mm culture dish, and adding a proper amount of sodium chloride injection to wash for 8 times. Transferring the washed Wharton jelly tissue into a 50ml centrifuge tube, and shearing the Wharton jelly tissue into a tissue with the volume of 1-2 mm by using ophthalmic scissors3Small pieces of (a). Sucking 0.5ml of Whistle's jelly tissue paste to 75cm with a Pasteur pipette2In the flask, the tissue was evenly spread with a 5ml pipette on the bottom of the flask.
And S3, placing the culture bottle in a biological safety cabinet for standing until all tissues adhere to the wall. Marking of adherence: the culture bottle is reversely buckled, the tissue cannot slip, and the tissue is in contact with the bottom of the bottle without water stain visible to naked eyes.
After the tissue is attached to the wall,5ml of primary culture medium is added into the culture bottle, and the bottle body is slowly shaken after the primary culture medium is added, so that the culture medium completely covers the bottom surface. Placing the culture flask at 37.0 deg.C and 5.0% CO2Culturing in an incubator.
S4, on day 5 after the tissue piece was inoculated, the cultured cells were taken out from the incubator. And (4) observing the growth condition of the cells under a microscope, and if spindle-shaped adherent cells appear around the tissue block, performing fluid infusion operation, and supplementing 5ml of primary culture medium into the culture flask. After the operation was completed, the flask was placed at 37.0 ℃ with 5.0% CO2And continuing culturing in the incubator.
S5, on day 8 after the tissue block was inoculated, the cultured cells were taken out from the incubator. The growth of the cells was observed under a microscope, and if it was found that more than 30% of the tissue pieces had cells on the edges, the medium was changed, and after the culture supernatant and the detached tissue pieces were removed by pipette, 10ml of the primary medium was added to the culture flask. After the operation was completed, the flask was placed at 37.0 ℃ with 5.0% CO2Continuously culturing in an incubator; when more than 50% of the tissue blocks are observed to climb out of the cells around the tissue blocks under a microscope and the local fusion degree is more than 80%, the primary culture is completed.
The primary culture medium at least comprises the following components in parts by weight: 100 parts of mesenchymal stem cell serum-free medium and 0.05 part of fibronectin.
The mesenchymal stem cell serum-free culture medium is purchased from Wuhan Punuo Sai Life technologies, Inc., with the product number of CM-SC 01;
the fibronectin was purchased from Shanghai leaf Biotech, Inc. under the trade designation S12073-5 mg.
The comparative example 4 also provides a primary cultured human umbilical cord mesenchymal stem cell which is obtained by the primary culture method of the stem cell with high cell adherence capability.
Evaluation of Performance
1. Wall time test
When the flasks were placed in a biosafety cabinet and left to stand in the above examples and comparative example S3, whether the tissues were completely attached to the wall was examined every 5 minutes, and the attachment time was recorded in Table 1.
2. Cell morphology testing
The human umbilical cord mesenchymal stem cells primarily cultured in examples 1 to 5 are prepared into working library cells, FIG. 1 is a morphology chart of the working library cells prepared from the human umbilical cord mesenchymal stem cells primarily cultured in example 1, and the morphology chart of the working library cells prepared from the human umbilical cord mesenchymal stem cells primarily cultured in examples 2 to 5 is similar to that of FIG. 1.
TABLE 1
Examples Adherence time/min
Example 1 25
Example 2 25
Example 3 25
Example 4 30
Example 5 30
Comparative example 1 60
Comparative example 2 55
Comparative example 3 40
Comparative example 4 45
According to the embodiment and the comparative example, the invention provides the primary culture method of the stem cells with high cell adherence capacity, the serum-free culture medium with definite components is used, the uncertainty of the components of the serum culture medium and the instability of serum culture are avoided, the cell adherence capacity in the serum-free culture medium is improved, the adherence time is reduced, the efficiency is improved, and the cells of the working bank prepared from the human umbilical cord mesenchymal stem cells subjected to primary culture have good morphology.
Finally, it should be understood that the above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A primary culture method of stem cells with high cell adherence capability is characterized in that: at least comprises the following steps:
s1, clipping the umbilical cord, transferring the umbilical cord into a culture dish, extruding residual blood and clipping; then transferring the tissue to a new culture dish to strip off the blood vessel and the adventitia to obtain a Wharton jelly tissue;
s2, placing the Wharton jelly tissue in a new culture dish, and adding sodium chloride injection for cleaning; transferring into a centrifuge tube, and cutting into small pieces; sucking Whitmania lacustra tissue paste and evenly spreading the Whitmania lacustra tissue paste at the bottom of the culture bottle;
s3, placing the culture bottle in a biological safety cabinet for standing, adding a primary culture medium into the culture bottle after the tissue adheres to the wall, and slowly shaking the bottle body to enable the culture medium to completely cover the bottom surface; placing the culture bottle into an incubator for culture.
2. The primary culture method of stem cells with high cell adherence capacity of claim 1, wherein: and S2, washing the sodium chloride injection for 8-10 times.
3. The primary culture method of stem cells with high cell adherence capacity according to claim 1, wherein the volume of the small block cut in S2 is 1-2 mm3。
4. The primary culture method of stem cells with high cell adherence capacity of claim 1, wherein: the culture conditions of the incubator are 37.0 ℃ and 5.0% CO2。
5. The primary culture method of stem cells with high cell adherence capacity of claim 1, wherein: the primary culture medium at least comprises the following components: serum-free medium of mesenchymal stem cells, fibronectin.
6. The primary culture method of stem cells with high cell adherence capacity of claim 5, wherein: the primary culture medium at least comprises the following components in parts by weight: 98-100 parts of mesenchymal stem cell serum-free medium and 0-0.5 part of fibronectin.
7. The primary culture method of stem cells with high cell adherence capacity of claim 6, wherein: the primary culture medium at least comprises the following components in parts by weight: 100 parts of mesenchymal stem cell serum-free medium and 0.25 part of fibronectin.
8. The primary culture method of stem cells with high cell adherence capacity according to any one of claims 1 to 7, further comprising: and (3) putting the culture bottle into an incubator for culturing for 4-6 days, supplementing a primary culture medium into the culture bottle, and continuing culturing.
9. The primary culture method of stem cells with high cell adherence capacity according to any one of claims 1 to 7, further comprising: after the culture bottle is placed into an incubator to be cultured for 7-9 days, a pipette is used for sucking and removing the supernatant fluid and the fallen tissue blocks of the culture bottle, and then primary culture medium is added for continuous culture; the amount of the primary medium added thereto was 2 times as much as the amount of the primary medium added in S1.
10. A primary cultured human umbilical cord mesenchymal stem cell, which is obtained by culturing the stem cell with high anchorage force according to the primary culture method of the stem cell of any one of claims 1 to 9.
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