1. A method for culturing adipose-derived stem cells is characterized by comprising the following steps:

step one, mixing the fat block and digestive juice according to the volume ratio of 2: (0.8-1.2), mixing, digesting for 10-90 min at 30-40 ℃ by shaking, standing, sucking the upper layer of the adipocyte liquid, adding the upper layer of the adipocyte liquid into a culture medium, sealing, centrifuging to remove supernatant, adding the culture medium, and blowing and beating cells to obtain the adipose-derived stem cell suspension for later use;

and step two, dropwise adding a culture medium to moisten the top of the culture bottle, inoculating the fat-derived stem cell suspension to the moistened top of the culture bottle, shaking the culture bottle to enable the fat-derived stem cell suspension to be in contact with the top of the culture bottle, turning the culture bottle over 180 degrees, paving the fat-derived stem cell suspension on the bottom surface of the culture bottle, and carrying out constant-temperature culture at 35-40 ℃.

2. The method of claim 1, wherein the method comprises: in the first step, the volume ratio of the fat block to the digestive juice is 2: 1.

3. the method for culturing the adipose-derived stem cells according to claim 2, wherein: the digestion solution in the first step contains collagenase type I with the concentration of 0.1-5 mg/ml.

4. The method of claim 1, wherein the method comprises: in the first step, the temperature of the shaking digestion is 35-39 ℃, and the time is 20-60 min.

5. The method for culturing the adipose-derived stem cells according to claim 4, wherein: in the first step, the temperature of the shaking digestion is 37 ℃, and the time is 20-60 min.

6. The method for culturing the adipose-derived stem cells according to any one of claims 1 to 5, wherein: the preparation process of the fat block in the first step comprises the following steps: cleaning adipose tissue with D-Hanks buffer solution, and cutting to obtain fat block.

7. The method of claim 1, wherein the method comprises: the culture medium contains compound growth factors.

8. The method of claim 1, wherein the method comprises: and the material of the culture bottle in the second step is oleophylic and hydrophobic material.

9. The method for culturing the adipose-derived stem cells according to claim 1 or 8, wherein: the temperature for constant temperature culture in the second step is 37 ℃.

Background

Adipose-derived stem cells (ADSCs) are a class of stem cells that can self-renew and propagate, and have the multipotentiality of common stem cells and stable in vitro multi-generation proliferation capacity. The collagen can be differentiated into fat cells, osteoblasts, chondrocytes, myocytes, nerve cells and the like under the action of different induction factors, can secrete a large amount of various cytokines and promote angiogenesis factors and apoptosis factors, can promote the fibroblasts to secrete type I collagen, type III collagen and fibronectin through paracrine action, promote collagen synthesis, and can play roles in resisting inflammation, oxidation, aging, injury repair and the like. In addition, the adipose tissue reserves are abundant and easy to obtain, and have no ethical dispute, and the method is one of ideal seed cell sources in tissue engineering research.

At present, the extraction mode of the adipose-derived stem cells mostly adopts a mechanical method, an enzyme digestion method and a tissue adherent culture method. The operation process of the enzyme digestion method and the mechanical method is complicated; in addition, the enzyme digestion method has long digestion time, and the components of the fat blood vessel matrix fragments obtained after digestion are complex. The tissue adherence method has the advantages of few operation steps, short experimental treatment time, no addition of exogenous collagenase, cell factors and the like, and low cost, but in the operation process, the fat oil drops block adherence of cells, so that the tissue blocks are difficult to adhere to the walls, and the tissue blocks are easy to float when liquid is changed, and the cell extraction efficiency is not high. In addition, the surface of a sterile disposable culture bottle made of polystyrene material used at ordinary times has strong oleophylic property, and the tissue adherence method is used for extracting the adipose-derived stem cells, so that the bottom of the culture bottle is inevitably stained with digested fat oil drops, and cells at the part stained with the fat oil drops cannot adhere to the wall to grow, so that the effective area at the bottom of the culture bottle is greatly reduced, and the grown cells are also greatly reduced.

Disclosure of Invention

In view of the above disadvantages of the prior art, the present invention aims to provide a method for culturing adipose-derived stem cells, which solves the problems of low cell attachment rate and low cell extraction efficiency caused by the inhibition of cell attachment by adipose oil droplets in the tissue attachment method in the prior art. According to the invention, by utilizing the oleophylic characteristic of the culture bottle and adsorbing the fat oil drops on the top in advance, the fat oil drops on the bottom surface of the culture bottle are avoided, and the cell adherence rate and the cell extraction efficiency are improved.

In order to attain the above and other related objects,

the invention provides a culture method of adipose-derived stem cells, which comprises the following steps:

step one, mixing the fat block and digestive juice according to the volume ratio of 2: (0.8-1.2), mixing, digesting for 10-90 min at 30-40 ℃ by shaking, standing, sucking the upper layer of the adipocyte liquid, adding the upper layer of the adipocyte liquid into a culture medium, sealing, centrifuging to remove supernatant, adding the culture medium, and blowing and beating cells to obtain the adipose-derived stem cell suspension for later use;

and step two, dropwise adding a culture medium to moisten the top of the culture bottle, inoculating the fat-derived stem cell suspension to the moistened top of the culture bottle, shaking the culture bottle to enable the fat-derived stem cell suspension to be in contact with the top of the culture bottle, turning the culture bottle over 180 degrees, paving the fat-derived stem cell suspension on the bottom surface of the culture bottle, and carrying out constant-temperature culture at 35-40 ℃.

Mixing the fat block and the digestive juice, shaking for digestion, transferring the upper layer of fat cell liquid into a culture medium for centrifugation, and blowing and beating to obtain the fat-derived stem cell suspension. Inoculating the fat-derived stem cell suspension on the top of a culture bottle which is wetted in advance, shaking the culture bottle to enable the fat-derived stem cell suspension to be in full contact with the top of the culture bottle, and enabling fat oil drops to be adsorbed on the top of the culture bottle through a shaking process by utilizing the oleophylic characteristic of the culture bottle. The fat oil drops are still adsorbed on the top of the culture bottle through 180-degree rotation, and the fat-derived stem cell suspension can be spread on the bottom surface of the culture bottle, so that the fat oil drops on the bottom surface of the culture bottle are avoided, and the cell adherence rate and the cell extraction efficiency are improved.

In an embodiment of the present invention, the volume ratio of the fat block to the digestive juice in the first step is 2: 1.

in an embodiment of the present invention, the digestion solution in the first step contains collagenase type I with a concentration of 0.1-5 mg/ml.

In an embodiment of the present invention, the temperature of the shaking digestion in the first step is 35 to 39 ℃ and the time is 20 to 60 min.

In an embodiment of the present invention, the temperature of the shaking digestion in the first step is 37 ℃ and the time is 20-60 min.

In an embodiment of the present invention, the process of making the fat block in the first step is as follows: cleaning adipose tissue with D-Hanks buffer solution, removing residual blood and tissue debris, and cutting adipose tissue into pieces to obtain fat block.

D-Hanks buffer A phenol red-free D-Hanks buffer was used.

In one embodiment of the present invention, the medium contains a complex growth factor.

In an embodiment of the present invention, the material of the culture flask in the second step is oleophilic and hydrophobic material.

The oleophilic and hydrophobic material can achieve the effect of absorbing fat oil drops, but a sterile disposable culture bottle made of polystyrene material is generally adopted.

The top and side surfaces of a sterile disposable culture flask made of polystyrene material are also made of the same material and also have surface oleophilic properties, which are not available in conventional cell culture. Therefore, by using the method, before the step of inoculating the adipose-derived stem cell suspension into the culture bottle is started, only a small amount of culture medium is added, the culture medium is used for wetting the top of the culture bottle, fat oil drops are more easily adsorbed by the top (namely the top surface) of the culture bottle, then the suspension to be inoculated is inoculated to the top of the wetted culture bottle, the top of the culture bottle is lightly shaken, the adipose-derived stem cell suspension is fully contacted with the top surface, then the culture bottle is turned over for 180 degrees, and the adipose-derived stem cell suspension almost without oil drops is spread on the bottom surface of the culture bottle for incubation and culture.

In one embodiment of the present invention, the temperature of the isothermal culture in the second step is 37 ℃.

As described above, the method for culturing adipose-derived stem cells according to the present invention has the following advantageous effects: mixing the fat block and the digestive juice, shaking for digestion, transferring the upper layer of fat cell liquid into a culture medium for centrifugation, and blowing and beating to obtain the fat-derived stem cell suspension. Inoculating the fat-derived stem cell suspension on the top of a culture bottle which is wetted in advance, shaking the culture bottle to enable the fat-derived stem cell suspension to be in full contact with the top of the culture bottle, and enabling fat oil drops to be adsorbed on the top of the culture bottle through a shaking process by utilizing the oleophylic characteristic of the culture bottle. The fat oil drops are still adsorbed on the top of the culture bottle through 180-degree rotation, and the fat-derived stem cell suspension can be spread on the bottom surface of the culture bottle, so that the fat oil drops on the bottom surface of the culture bottle are avoided, and the cell adherence rate and the cell extraction efficiency are improved.

Drawings

In FIG. 1, A is the cell distribution diagram of example 1 at 400 times magnification, and B is the cell distribution diagram of comparative example 1 at 400 times magnification.

In FIG. 2, A is the cell distribution diagram of example 2 at 400 times magnification, and B is the cell distribution diagram of comparative example 2 at 400 times magnification.

In FIG. 3, A is the cell distribution diagram of example 3 at 400 times magnification, and B is the cell distribution diagram of comparative example 3 at 400 times magnification.

In FIG. 4, A is the cell distribution diagram of example 4 at 400 times magnification, and B is the cell distribution diagram of comparative example 4 at 400 times magnification.

In FIG. 5, A is the cell distribution diagram of example 5 at 400 times magnification, and B is the cell distribution diagram of comparative example 5 at 400 times magnification.

In FIG. 6, A is the cell distribution diagram of example 6 at 400 times magnification, and B is the cell distribution diagram of comparative example 6 at 400 times magnification.

In FIG. 7, A is the cell distribution diagram of example 7 at 400 times magnification, and B is the cell distribution diagram of comparative example 7 at 400 times magnification.

In FIG. 8, A is the cell distribution diagram of example 8 at 400 times magnification, and B is the cell distribution diagram of comparative example 8 at 400 times magnification.

In FIG. 9, A is the cell distribution diagram of example 9 at 400 times magnification, and B is the cell distribution diagram of comparative example 9 at 400 times magnification.

In FIG. 10, A is the cell distribution diagram of example 10 at 400 times magnification, and B is the cell distribution diagram of comparative example 10 at 400 times magnification.

FIG. 11 is a graph showing the cell distribution at 400 times magnification after the culture for 5d in example 5, and B shows the cell distribution at 400 times magnification after the culture for 5d in comparative example 5.

In FIG. 12, A is the cell distribution map at 400 times magnification after 6d of culture in example 5, and B is the cell distribution map at 400 times magnification after 6d of culture in comparative example 5.

In FIG. 13, A is the cell distribution map at 400 times magnification after 8d of culture in example 5, and B is the cell distribution map at 400 times magnification after 8d of culture in comparative example 5.

In FIG. 14, A is the cell distribution map at 400 times magnification after the culture for 11d in example 5, and B is the cell distribution map at 400 times magnification after the culture for 11d in comparative example 5.

In FIG. 15, A is the cell distribution map at 400 times magnification after the culture for 5d of example 9, and B is the cell distribution map at 400 times magnification after the culture for 5d of comparative example 9.

In FIG. 16, A is the cell distribution map at 400 times magnification after 6d of culture in example 9, and B is the cell distribution map at 400 times magnification after 6d of culture in comparative example 9.

In FIG. 17, A is the cell distribution map at 400 times magnification after 9d culture of example 9, and B is the cell distribution map at 400 times magnification after 9d culture of comparative example 9.

FIG. 18 is a bar graph showing the mean number of example cells and the mean number of comparative example cells at 6d, 8d, and 9 d.

FIG. 19 is a view showing the inner surface of the flask of comparative example 1 taken under an inverted microscope.

FIG. 20 is a view showing the inner surface of the flask of comparative example 2 taken under an inverted microscope.

FIG. 21 is a view showing the inner surface of the flask of comparative example 3 taken under an inverted microscope.

Detailed Description

The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will become apparent to those skilled in the art from the present disclosure.

Example 1

A method for culturing adipose-derived stem cells comprises the following steps:

step one, mixing the fat block and digestive juice according to the volume ratio of 2: 0.8, mixing, wherein the digestive juice contains type I collagenase with the concentration of 0.5mg/ml, shaking and digesting for 30min at the temperature of 35 ℃, standing, sucking the upper layer of the adipocyte juice, adding the adipocyte juice into a culture medium, sealing, centrifuging to remove supernatant, adding the culture medium, and blowing and beating cells to obtain the adipogenic stem cell suspension for later use;

and step two, dropwise adding a culture medium to wet the top of the culture bottle (polystyrene material), inoculating the fat-derived stem cell suspension to the top of the wetted culture bottle, shaking the culture bottle to enable the fat-derived stem cell suspension to be in contact with the top of the culture bottle, turning over the culture bottle by 180 degrees to enable the fat-derived stem cell suspension to be paved on the bottom surface of the culture bottle, and carrying out constant-temperature culture at 35 ℃. Periodically (3d) medium was changed until the adipose stem cells were confluent at the bottom of the flask.

Specifically, the adipose tissues are washed by D-Hanks buffer solution without phenol red, residual blood and tissue fragments are removed, and then the adipose tissues are cut into pieces to obtain the fat blocks.

Specifically, the culture medium contains a compound growth factor.

Comparative example 1

A method for culturing adipose-derived stem cells comprises the following steps:

step one, mixing the fat block and digestive juice according to the volume ratio of 2: 0.8, mixing, wherein the digestive juice contains type I collagenase with the concentration of 0.5mg/ml, shaking and digesting for 30min at the temperature of 35 ℃, standing, sucking the upper layer of the adipocyte juice, adding the adipocyte juice into a culture medium, sealing, centrifuging to remove supernatant, adding the culture medium, and blowing and beating cells to obtain the adipogenic stem cell suspension for later use;

and step two, dropwise adding a culture medium to wet the bottom of the culture bottle (polystyrene material), inoculating the fat-derived stem cell suspension to the bottom of the wetted culture bottle, and performing constant-temperature culture at 35 ℃. Periodically (3d) medium was changed until the adipose stem cells were confluent at the bottom of the flask.

Specifically, the adipose tissues are washed by D-Hanks buffer solution without phenol red, residual blood and tissue fragments are removed, and then the adipose tissues are cut into pieces to obtain the fat blocks.

Specifically, the culture medium contains a compound growth factor.

Example 2

A method for culturing adipose-derived stem cells comprises the following steps:

step one, mixing the fat block and digestive juice according to the volume ratio of 2: 0.9, mixing, wherein the digestive juice contains 1mg/ml type I collagenase, shaking and digesting for 40min at 36 ℃, standing, sucking the upper layer of the adipocyte juice, adding the adipocyte juice into a culture medium, sealing, centrifuging to remove supernatant, adding the culture medium, and blowing and beating cells to obtain the adipogenic stem cell suspension for later use;

and step two, dropwise adding a culture medium to wet the top of the culture bottle (polystyrene material), inoculating the fat-derived stem cell suspension to the top of the wetted culture bottle, shaking the culture bottle to enable the fat-derived stem cell suspension to be in contact with the top of the culture bottle, turning the culture bottle by 180 degrees, paving the fat-derived stem cell suspension on the bottom of the culture bottle, and carrying out constant-temperature culture at 35 ℃. Periodically (3d) medium was changed until the adipose stem cells were confluent at the bottom of the flask.

Specifically, the adipose tissues are washed by D-Hanks buffer solution without phenol red, residual blood and tissue fragments are removed, and then the adipose tissues are cut into pieces to obtain the fat blocks.

Specifically, the culture medium contains a compound growth factor.

Comparative example 2

A method for culturing adipose-derived stem cells comprises the following steps:

step one, mixing the fat block and digestive juice according to the volume ratio of 2: 0.9, mixing, wherein the digestive juice contains 1mg/ml type I collagenase, shaking and digesting for 40min at 36 ℃, standing, sucking the upper layer of the adipocyte juice, adding the adipocyte juice into a culture medium, sealing, centrifuging to remove supernatant, adding the culture medium, and blowing and beating cells to obtain the adipogenic stem cell suspension for later use;

and step two, dropwise adding a culture medium to wet the bottom of the culture bottle (polystyrene material), inoculating the fat-derived stem cell suspension to the bottom of the wetted culture bottle, and performing constant-temperature culture at 35 ℃. Periodically (3d) medium was changed until the adipose stem cells were confluent at the bottom of the flask.

Example 3

A method for culturing adipose-derived stem cells comprises the following steps:

step one, mixing the fat block and digestive juice according to the volume ratio of 2: 1.2, mixing, wherein the digestive juice contains type I collagenase with the concentration of 3mg/ml, shaking and digesting for 60min at 38 ℃, standing, sucking the upper layer of adipocyte juice, adding the adipocyte juice into a culture medium, sealing, centrifuging to remove supernatant, adding the culture medium, and blowing and beating cells to obtain the adipogenic stem cell suspension for later use;

and step two, dropwise adding a culture medium to wet the top of the culture bottle (polystyrene material), inoculating the fat-derived stem cell suspension to the top of the wetted culture bottle, shaking the culture bottle to enable the fat-derived stem cell suspension to be in contact with the top of the culture bottle, turning the culture bottle by 180 degrees, paving the fat-derived stem cell suspension on the bottom of the culture bottle, and carrying out constant-temperature culture at 38 ℃. Periodically (3d) medium was changed until the adipose stem cells were confluent at the bottom of the flask.

Specifically, the adipose tissues are washed by D-Hanks buffer solution without phenol red, residual blood and tissue fragments are removed, and then the adipose tissues are cut into pieces to obtain the fat blocks.

Specifically, the culture medium contains a compound growth factor.

Comparative example 3

A method for culturing adipose-derived stem cells comprises the following steps:

step one, mixing the fat block and digestive juice according to the volume ratio of 2: 1.2, mixing, wherein the digestive juice contains type I collagenase with the concentration of 3mg/ml, shaking and digesting for 60min at 38 ℃, standing, sucking the upper layer of adipocyte juice, adding the adipocyte juice into a culture medium, sealing, centrifuging to remove supernatant, adding the culture medium, and blowing and beating cells to obtain the adipogenic stem cell suspension for later use;

and step two, dropwise adding a culture medium to wet the bottom of the culture bottle (polystyrene material), inoculating the fat-derived stem cell suspension to the bottom of the wetted culture bottle, and performing constant-temperature culture at 38 ℃. Periodically (3d) medium was changed until the adipose stem cells were confluent at the bottom of the flask.

Specifically, the adipose tissues are washed by D-Hanks buffer solution without phenol red, residual blood and tissue fragments are removed, and then the adipose tissues are cut into pieces to obtain the fat blocks.

Specifically, the culture medium contains a compound growth factor.

Example 4

A method for culturing adipose-derived stem cells comprises the following steps:

step one, mixing the fat block and digestive juice according to the volume ratio of 2: 1.1, mixing, wherein the digestive juice contains type I collagenase with the concentration of 4mg/ml, shaking and digesting for 30min at 39 ℃, standing, sucking the upper layer of adipocyte juice, adding the adipocyte juice into a culture medium, sealing, centrifuging to remove supernatant, adding the culture medium, and blowing and beating cells to obtain the adipogenic stem cell suspension for later use;

and step two, dropwise adding a culture medium to wet the top of the culture bottle (polystyrene material), inoculating the fat-derived stem cell suspension to the top of the wetted culture bottle, shaking the culture bottle to enable the fat-derived stem cell suspension to be in contact with the top of the culture bottle, turning the culture bottle by 180 degrees, paving the fat-derived stem cell suspension on the bottom of the culture bottle, and carrying out constant-temperature culture at 39 ℃. Periodically (3d) medium was changed until the adipose stem cells were confluent at the bottom of the flask.

Specifically, the adipose tissues are washed by D-Hanks buffer solution without phenol red, residual blood and tissue fragments are removed, and then the adipose tissues are cut into pieces to obtain the fat blocks.

Specifically, the culture medium contains a compound growth factor.

Comparative example 4

A method for culturing adipose-derived stem cells comprises the following steps:

step one, mixing the fat block and digestive juice according to the volume ratio of 2: 1.1, mixing, wherein the digestive juice contains type I collagenase with the concentration of 4mg/ml, shaking and digesting for 30min at 39 ℃, standing, sucking the upper layer of adipocyte juice, adding the adipocyte juice into a culture medium, sealing, centrifuging to remove supernatant, adding the culture medium, and blowing and beating cells to obtain the adipogenic stem cell suspension for later use;

and step two, dropwise adding a culture medium to wet the bottom of the culture bottle (polystyrene material), inoculating the fat-derived stem cell suspension to the bottom of the wetted culture bottle, and performing constant-temperature culture at 39 ℃. Periodically (3d) medium was changed until the adipose stem cells were confluent at the bottom of the flask.

Specifically, the adipose tissues are washed by D-Hanks buffer solution without phenol red, residual blood and tissue fragments are removed, and then the adipose tissues are cut into pieces to obtain the fat blocks.

Specifically, the culture medium contains a compound growth factor.

Example 5

A method for culturing adipose-derived stem cells comprises the following steps:

step one, mixing the fat block and digestive juice according to the volume ratio of 2: 1.1, mixing, wherein the digestive juice contains type I collagenase with the concentration of 3mg/ml, shaking and digesting for 40min at 38 ℃, standing, sucking the upper layer of adipocyte juice, adding the adipocyte juice into a culture medium, sealing, centrifuging to remove supernatant, adding the culture medium, and blowing and beating cells to obtain the adipogenic stem cell suspension for later use;

and step two, dropwise adding a culture medium to wet the top of the culture bottle (polystyrene material), inoculating the fat-derived stem cell suspension to the top of the wetted culture bottle, shaking the culture bottle to enable the fat-derived stem cell suspension to be in contact with the top of the culture bottle, turning the culture bottle by 180 degrees, paving the fat-derived stem cell suspension on the bottom of the culture bottle, and carrying out constant-temperature culture at 38 ℃. Periodically (3d) medium was changed until the adipose stem cells were confluent at the bottom of the flask.

Specifically, the adipose tissues are washed by D-Hanks buffer solution without phenol red, residual blood and tissue fragments are removed, and then the adipose tissues are cut into pieces to obtain the fat blocks.

Specifically, the culture medium contains a compound growth factor.

Comparative example 5

A method for culturing adipose-derived stem cells comprises the following steps:

step one, mixing the fat block and digestive juice according to the volume ratio of 2: 1.1, mixing, wherein the digestive juice contains type I collagenase with the concentration of 3mg/ml, shaking and digesting for 40min at 38 ℃, standing, sucking the upper layer of adipocyte juice, adding the adipocyte juice into a culture medium, sealing, centrifuging to remove supernatant, adding the culture medium, and blowing and beating cells to obtain the adipogenic stem cell suspension for later use;

and step two, dropwise adding a culture medium to wet the bottom of the culture bottle (polystyrene material), inoculating the fat-derived stem cell suspension to the bottom of the wetted culture bottle, and performing constant-temperature culture at 38 ℃. Periodically (3d) medium was changed until the adipose stem cells were confluent at the bottom of the flask.

Specifically, the adipose tissues are washed by D-Hanks buffer solution without phenol red, residual blood and tissue fragments are removed, and then the adipose tissues are cut into pieces to obtain the fat blocks.

Specifically, the culture medium contains a compound growth factor.

Example 6

A method for culturing adipose-derived stem cells comprises the following steps:

step one, mixing the fat block and digestive juice according to the volume ratio of 2: 1.2, mixing, wherein the digestive juice contains type I collagenase with the concentration of 5mg/ml, shaking and digesting for 60min at 32 ℃, standing, sucking the upper layer of the adipocyte juice, adding the adipocyte juice into a culture medium, sealing, centrifuging to remove supernatant, adding the culture medium, and blowing and beating cells to obtain the adipogenic stem cell suspension for later use;

and step two, dropwise adding a culture medium to wet the top of the culture bottle (polystyrene material), inoculating the fat-derived stem cell suspension to the top of the wetted culture bottle, shaking the culture bottle to enable the fat-derived stem cell suspension to be in contact with the top of the culture bottle, turning the culture bottle by 180 degrees, paving the fat-derived stem cell suspension on the bottom of the culture bottle, and carrying out constant-temperature culture at 35 ℃. Periodically (3d) medium was changed until the adipose stem cells were confluent at the bottom of the flask.

Specifically, the adipose tissues are washed by D-Hanks buffer solution without phenol red, residual blood and tissue fragments are removed, and then the adipose tissues are cut into pieces to obtain the fat blocks.

Specifically, the culture medium contains a compound growth factor.

Comparative example 6

A method for culturing adipose-derived stem cells comprises the following steps:

step one, mixing the fat block and digestive juice according to the volume ratio of 2: 1.2, mixing, wherein the digestive juice contains type I collagenase with the concentration of 5mg/ml, shaking and digesting for 60min at 32 ℃, standing, sucking the upper layer of the adipocyte juice, adding the adipocyte juice into a culture medium, sealing, centrifuging to remove supernatant, adding the culture medium, and blowing and beating cells to obtain the adipogenic stem cell suspension for later use;

and step two, dropwise adding a culture medium to wet the bottom of the culture bottle (polystyrene material), inoculating the fat-derived stem cell suspension to the bottom of the wetted culture bottle, and performing constant-temperature culture at 35 ℃. Periodically (3d) medium was changed until the adipose stem cells were confluent at the bottom of the flask.

Specifically, the adipose tissues are washed by D-Hanks buffer solution without phenol red, residual blood and tissue fragments are removed, and then the adipose tissues are cut into pieces to obtain the fat blocks.

Specifically, the culture medium contains a compound growth factor.

Example 7

A method for culturing adipose-derived stem cells comprises the following steps:

step one, mixing the fat block and digestive juice according to the volume ratio of 2: 1, mixing, wherein the digestive juice contains type I collagenase with the concentration of 3mg/ml, shaking and digesting for 60min at 34 ℃, standing, sucking the upper layer of adipocyte juice, adding the adipocyte juice into a culture medium, sealing, centrifuging to remove supernatant, adding the culture medium, and blowing and beating cells to obtain the adipogenic stem cell suspension for later use;

and step two, dropwise adding a culture medium to wet the top of the culture bottle (polystyrene material), inoculating the fat-derived stem cell suspension to the top of the wetted culture bottle, shaking the culture bottle to enable the fat-derived stem cell suspension to be in contact with the top of the culture bottle, turning the culture bottle by 180 degrees, paving the fat-derived stem cell suspension on the bottom of the culture bottle, and carrying out constant-temperature culture at 38 ℃. Periodically (3d) medium was changed until the adipose stem cells were confluent at the bottom of the flask.

Specifically, the adipose tissues are washed by D-Hanks buffer solution without phenol red, residual blood and tissue fragments are removed, and then the adipose tissues are cut into pieces to obtain the fat blocks.

Specifically, the culture medium contains a compound growth factor.

Comparative example 7

A method for culturing adipose-derived stem cells comprises the following steps:

step one, mixing the fat block and digestive juice according to the volume ratio of 2: 1, mixing, wherein the digestive juice contains type I collagenase with the concentration of 3mg/ml, shaking and digesting for 60min at 34 ℃, standing, sucking the upper layer of adipocyte juice, adding the adipocyte juice into a culture medium, sealing, centrifuging to remove supernatant, adding the culture medium, and blowing and beating cells to obtain the adipogenic stem cell suspension for later use;

and step two, dropwise adding a culture medium to wet the bottom of the culture bottle (polystyrene material), inoculating the fat-derived stem cell suspension to the bottom of the wetted culture bottle, and performing constant-temperature culture at 38 ℃. Periodically (3d) medium was changed until the adipose stem cells were confluent at the bottom of the flask.

Specifically, the adipose tissues are washed by D-Hanks buffer solution without phenol red, residual blood and tissue fragments are removed, and then the adipose tissues are cut into pieces to obtain the fat blocks.

Specifically, the culture medium contains a compound growth factor.

Example 8

A method for culturing adipose-derived stem cells comprises the following steps:

step one, mixing the fat block and digestive juice according to the volume ratio of 2: 1, mixing, wherein the digestive juice contains type I collagenase with the concentration of 2mg/ml, shaking and digesting for 20min at 38 ℃, standing, sucking the upper layer of adipocyte juice, adding the adipocyte juice into a culture medium, sealing, centrifuging to remove supernatant, adding the culture medium, and blowing and beating cells to obtain the adipose-derived stem cell suspension for later use;

and step two, dropwise adding a culture medium to wet the top of the culture bottle (polystyrene material), inoculating the fat-derived stem cell suspension to the top of the wetted culture bottle, shaking the culture bottle to enable the fat-derived stem cell suspension to be in contact with the top of the culture bottle, turning the culture bottle by 180 degrees, paving the fat-derived stem cell suspension on the bottom of the culture bottle, and carrying out constant-temperature culture at 38 ℃. Periodically (3d) medium was changed until the adipose stem cells were confluent at the bottom of the flask.

Specifically, the adipose tissues are washed by D-Hanks buffer solution without phenol red, residual blood and tissue fragments are removed, and then the adipose tissues are cut into pieces to obtain the fat blocks.

Specifically, the culture medium contains a compound growth factor.

Comparative example 8

A method for culturing adipose-derived stem cells comprises the following steps:

step one, mixing the fat block and digestive juice according to the volume ratio of 2: 1, mixing, wherein the digestive juice contains type I collagenase with the concentration of 2mg/ml, shaking and digesting for 20min at 38 ℃, standing, sucking the upper layer of adipocyte juice, adding the adipocyte juice into a culture medium, sealing, centrifuging to remove supernatant, adding the culture medium, and blowing and beating cells to obtain the adipose-derived stem cell suspension for later use;

and step two, dropwise adding a culture medium to wet the bottom of the culture bottle (polystyrene material), inoculating the fat-derived stem cell suspension to the bottom of the wetted culture bottle, and performing constant-temperature culture at 38 ℃. Periodically (3d) medium was changed until the adipose stem cells were confluent at the bottom of the flask.

Specifically, the adipose tissues are washed by D-Hanks buffer solution without phenol red, residual blood and tissue fragments are removed, and then the adipose tissues are cut into pieces to obtain the fat blocks.

Specifically, the culture medium contains a compound growth factor.

Example 9

A method for culturing adipose-derived stem cells comprises the following steps:

step one, mixing the fat block and digestive juice according to the volume ratio of 2: 1, mixing, wherein the digestive juice contains type I collagenase with the concentration of 2mg/ml, shaking and digesting for 45min at 37 ℃, standing, sucking the upper layer of adipocyte juice, adding the adipocyte juice into a culture medium, sealing, centrifuging to remove supernatant, adding the culture medium, and blowing and beating cells to obtain the adipogenic stem cell suspension for later use;

and step two, dropwise adding a culture medium to wet the top of the culture bottle (polystyrene material), inoculating the fat-derived stem cell suspension to the top of the wetted culture bottle, shaking the culture bottle to enable the fat-derived stem cell suspension to be in contact with the top of the culture bottle, turning the culture bottle by 180 degrees, paving the fat-derived stem cell suspension on the bottom of the culture bottle, and carrying out constant-temperature culture at 37 ℃. Periodically (3d) medium was changed until the adipose stem cells were confluent at the bottom of the flask.

Specifically, the adipose tissues are washed by D-Hanks buffer solution without phenol red, residual blood and tissue fragments are removed, and then the adipose tissues are cut into pieces to obtain the fat blocks.

Specifically, the culture medium contains a compound growth factor.

Comparative example 9

A method for culturing adipose-derived stem cells comprises the following steps:

step one, mixing the fat block and digestive juice according to the volume ratio of 2: 1, mixing, wherein the digestive juice contains type I collagenase with the concentration of 2mg/ml, shaking and digesting for 45min at 37 ℃, standing, sucking the upper layer of adipocyte juice, adding the adipocyte juice into a culture medium, sealing, centrifuging to remove supernatant, adding the culture medium, and blowing and beating cells to obtain the adipogenic stem cell suspension for later use;

and step two, dropwise adding a culture medium to wet the bottom of the culture bottle (polystyrene material), inoculating the fat-derived stem cell suspension to the bottom of the wetted culture bottle, and performing constant-temperature culture at 37 ℃. Periodically (3d) medium was changed until the adipose stem cells were confluent at the bottom of the flask.

Specifically, the adipose tissues are washed by D-Hanks buffer solution without phenol red, residual blood and tissue fragments are removed, and then the adipose tissues are cut into pieces to obtain the fat blocks.

Specifically, the culture medium contains a compound growth factor.

Example 10

A method for culturing adipose-derived stem cells comprises the following steps:

step one, mixing the fat block and digestive juice according to the volume ratio of 2: 1, mixing, wherein the digestive juice contains 1mg/ml type I collagenase, shaking and digesting for 30min at 37 ℃, standing, sucking the upper layer of adipocyte juice, adding the upper layer of adipocyte juice into a culture medium, sealing, centrifuging to remove supernatant, adding the culture medium, and blowing and beating cells to obtain the adipose-derived stem cell suspension for later use;

and step two, dropwise adding a culture medium to wet the top of the culture bottle (polystyrene material), inoculating the fat-derived stem cell suspension to the top of the wetted culture bottle, shaking the culture bottle to enable the fat-derived stem cell suspension to be in contact with the top of the culture bottle, turning the culture bottle by 180 degrees, paving the fat-derived stem cell suspension on the bottom of the culture bottle, and carrying out constant-temperature culture at 37 ℃. Periodically (3d) medium was changed until the adipose stem cells were confluent at the bottom of the flask.

Specifically, the adipose tissues are washed by D-Hanks buffer solution without phenol red, residual blood and tissue fragments are removed, and then the adipose tissues are cut into pieces to obtain the fat blocks.

Specifically, the culture medium contains a compound growth factor.

Comparative example 10

A method for culturing adipose-derived stem cells comprises the following steps:

step one, mixing the fat block and digestive juice according to the volume ratio of 2: 1, mixing, wherein the digestive juice contains 1mg/ml type I collagenase, shaking and digesting for 30min at 37 ℃, standing, sucking the upper layer of adipocyte juice, adding the upper layer of adipocyte juice into a culture medium, sealing, centrifuging to remove supernatant, adding the culture medium, and blowing and beating cells to obtain the adipose-derived stem cell suspension for later use;

and step two, dropwise adding a culture medium to wet the bottom of the culture bottle (polystyrene material), inoculating the fat-derived stem cell suspension to the bottom of the wetted culture bottle, and performing constant-temperature culture at 37 ℃. Periodically (3d) medium was changed until the adipose stem cells were confluent at the bottom of the flask.

Specifically, the adipose tissues are washed by D-Hanks buffer solution without phenol red, residual blood and tissue fragments are removed, and then the adipose tissues are cut into pieces to obtain the fat blocks.

Specifically, the culture medium contains a compound growth factor.

The number of cells collected over a certain period of time from the cells of examples 1 to 10 and corresponding comparative examples 1 to 10 is shown in table 1. The cell number is measured by combining a microscope with a blood counting chamber, cells are digested and collected by pancreatin to prepare cell suspension, the concentration of the cell suspension is measured by using the blood counting chamber, and the product of the concentration and the volume is the total number of the cells.

Table 1

As can be seen from the data in Table 1, the number of cells for each example was greater than the corresponding comparative example. Therefore, by utilizing the oleophylic characteristic of the culture bottle and adsorbing the fat oil drops on the top in advance, the cell adherence rate and the cell extraction efficiency can be improved.

The cells of examples 1 to 10 and corresponding comparative examples 1 to 10 were observed under a microscope. Each example and the corresponding comparative example were sampled at the same time to obtain the pictures of fig. 1 to 10 at 400 times magnification. As can be seen from fig. 1 to 10, each example had a larger number of cells than the corresponding comparative example under a microscope of the same magnification. Therefore, the characteristic of oleophylicity of the culture bottle is utilized, and the cell adherence rate and the cell extraction efficiency can be improved by adsorbing the fat oil drops on the top in advance.

The cells of example 5 and comparative example 5 were cultured in 5d, 6d, 8d, and 11d, respectively, and observed under a microscope, and the pictures of FIGS. 11 to 14 were obtained at 400-fold magnification. As can be seen from FIGS. 11 to 14, the number of cells in example 5 was larger than that in comparative example 5 under the same culture time and the same magnification.

The cells of example 9 and comparative example 9 were cultured for 5d, 6d, and 9d, respectively, and observed under a microscope, and the pictures of fig. 15 to 17 were obtained at 400-fold magnification. As can be seen from FIGS. 15 to 17, the number of cells in example 9 was larger than that in comparative example 9 under the same culture time and the same magnification.

The cell numbers of 6d, 8d and 9d cultured in the embodiments 1 to 10 are respectively measured, and the cell numbers of the embodiments 1 to 10 corresponding to the time periods are added and averaged to obtain the cell number average values of the embodiments corresponding to 6d, 8d and 9 d. And (3) respectively measuring the cell numbers of the cells cultured in the comparative examples 1 to 10 for 6d, 8d and 9d, and adding and averaging the cell numbers of the comparative examples 1 to 10 in corresponding time periods to obtain the average value of the cell numbers of the comparative examples corresponding to 6d, 8d and 9 d. As can be seen from fig. 18, the mean number of cells of the examples and the mean number of cells of the comparative examples were significantly different at 6d, 8d and 9 d.

The inner surfaces of the culture bottles of comparative example 1, comparative example 2 and comparative example 3 were photographed under an inverted microscope respectively in the culture process and are marked as fig. 19 to 21, and it can be seen in fig. 19 to 21 that the obvious fat oil drops stain the inner surface of the culture bottles to make the cells not grow there.

In conclusion, the adipose-derived stem cell suspension is inoculated on the top of the culture bottle which is wetted in advance, the culture bottle is shaken to enable the adipose-derived stem cell suspension to be in full contact with the top of the culture bottle, and fat oil drops are adsorbed on the top of the culture bottle through the shaking process by utilizing the oleophylic characteristic of the culture bottle. The fat oil drops are still adsorbed on the top of the culture bottle through 180-degree rotation, and the fat-derived stem cell suspension can be spread on the bottom surface of the culture bottle, so that the fat oil drops on the bottom surface of the culture bottle are avoided, and the cell adherence rate and the cell extraction efficiency are improved. Therefore, the invention effectively overcomes various defects in the prior art and has high industrial utilization value.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.