阅读说明：本技术 一种体外激活脂肪干细胞转化成原软骨细胞的方法 (Method for in vitro activating adipose-derived stem cells to convert adipose-derived stem cells into protochondrocytic cells ) 是由 谢海涛 薛卫巍 王斌 于 2019-11-20 设计创作，主要内容包括：本发明公开了一种体外激活脂肪干细胞转化成原软骨细胞的方法,包括如下步骤：配置DMEM培养基,所述DMEM培养基配比为1％胎牛血清、10ng/mlTGF－β、100ng/mlBMP－6、6.25μg/ml胰岛素、0.1μmol/L地塞米松、6.25μg/ml转铁蛋白、50μmol/L抗坏血酸磷酸酯；将形成微团的脂肪源干细胞置于DMEM培养基中进行培养；将培养7-9天的脂肪源干细胞注射进入关节腔转化为原软骨细胞。本发明具有节约时间、过程控制简单、产生的软骨细胞排斥低的优点。(The invention discloses a method for activating adipose-derived stem cells to convert into protochondrocytes in vitro, which comprises the following steps: preparing a DMEM medium, wherein the proportion of the DMEM medium is 1% fetal calf serum, 10ng/ml TGF-beta, 100ng/ml BMP-6, 6.25 mu g/ml insulin, 0.1 mu mol/L dexamethasone, 6.25 mu g/ml transferrin and 50 mu mol/L ascorbic acid phosphate; placing the adipose-derived stem cells forming the micelles in a DMEM culture medium for culturing; injecting the adipose-derived stem cells cultured for 7-9 days into joint cavity to transform into protochondrocytes. The invention has the advantages of time saving, simple process control and low rejection of the generated chondrocytes.)

1. A method for activating the transformation of adipose-derived stem cells into protochondrocytes in vitro, which comprises the following steps:

preparing a DMEM medium, wherein the proportion of the DMEM medium is 1% fetal calf serum, 10ng/ml TGF-beta, 100ng/ml BMP-6, 6.25 mu g/ml insulin, 0.1 mu mol/L dexamethasone, 6.25 mu g/ml transferrin and 50 mu mol/L ascorbic acid phosphate;

placing the adipose-derived stem cells forming the micelles in a DMEM culture medium for culturing;

injecting the adipose-derived stem cells cultured for 7-9 days into joint cavity to transform into protochondrocytes.

2. The method of claim 1, wherein: the preparation steps of the adipose-derived stem cells are as follows:

cleaning adipose tissues by using a buffer solution to remove residual blood and tissue fragments, cutting the cleaned adipose tissues into small pieces, digesting the small pieces in a shaking box for 20-60 min, standing the small pieces until the small pieces are layered, sucking upper layer adipose cell fluid, placing the adipose cell fluid in a box body at the temperature of 0-5 ℃ for culturing for 1-2 hours, sealing and centrifugally separating the box body, and removing supernatant to prepare adipose stem cell suspension;

placing the adipose-derived stem cell suspension in a cell activation medium for activation culture for 10-20 hours so as to obtain preliminarily expanded adipose-derived stem cells;

placing the adipose-derived stem cells into at least one cell culture medium and adding an activating reagent to perform adipose-derived stem cell suspension culture for 12-15 hours so as to obtain activated adipose-derived stem cells;

taking out the activated adipose-derived stem cells from the culture medium, adding normal saline, cleaning, adding enzyme solution for digestion, and culturing for three to six generations after the adipose-derived stem cells grow to 80-90% and are fused;

and naturally placing the culture solution after passage for 3-5 hours to obtain the micelle adipose-derived stem cells.

3. The method of claim 1, wherein: the adipose-derived stem cells cultured for 8 days are injected into the joint cavity and transformed into the original chondrocytes.

4. The method of claim 2, wherein: after the adipose-derived stem cells grow to 80% -90% and fuse, the cells are cultured for five generations.

5. The method of claim 2, wherein: the cell activation culture medium is a serum-free lymphocyte culture medium added with interleukin-2 and saperin.

6. The method of claim 2, wherein: the cell activation medium is supplemented with mammalian serum.

7. The method of claim 7, wherein: the mammalian serum is mammalian fetal serum.

8. The method of claim 2, wherein: the activating agent is a calcium ionophore.

9. The method of claim 9, wherein: the calcium ionophore is selected from A23187 or ionomycin.

Technical Field

The invention relates to the field of medicines, in particular to a method for activating adipose-derived stem cells to convert into protochondrocytes in vitro.

Background

Bone marrow mesenchymal stem cells (BMSCs) among Mesenchymal Stem Cells (MSCs) which have been used more so far are hot spots of research and widely applied to scientific research. In 2001, Zuk et al isolated 1 st from liposuction extracted adipose tissue suspensions to obtain multipotential stem cells. Adipose-derived stem cells (ASCs) are superior to BMSCs in that they are easily obtained, have a large quantity of materials, can be repeatedly obtained, and can proliferate rapidly. Meanwhile, ASCs can be differentiated into chondrocytes, osteoblasts, adipocytes, nerve cells, etc., respectively, in a specific microenvironment. Therefore, the ASCs are expected to become good tissue engineering seed cells.

The major problems are that (1) the currently known ASCs have a plurality of surface molecules, but most of the ASCs are crossed with other mesenchymal stem cells, and have no specific surface antigen, so that the combined screening by selecting the high-specificity surface marker antigen is difficult; (2) although many laboratories have attempted to induce differentiation of ASCs into cartilage tissue using different materials and techniques, none have avoided the consequences of inflammatory reactions between cartilage tissues; (3) in many experiments, the phenomenon of hypertrophy and hyperplasia of chondrocytes appears in the cultured chondrocytes in the later experiment results; (4) at present, the differentiation of the ASCs into the chondrocytes is basically realized by constructing a 3D model, but the clinical practicability of the method is not high.

Disclosure of Invention

In view of the above, the present invention provides a method for activating the transformation of adipose derived stem cells into chondroblasts in vitro.

The purpose of the invention is realized by adopting the following technical scheme:

a method for activating the transformation of adipose-derived stem cells into protochondrocytes in vitro, comprising the following steps:

preparing a DMEM medium, wherein the proportion of the DMEM medium is 1% fetal calf serum, 10ng/ml TGF-beta, 100ng/ml BMP-6, 6.25 mu g/ml insulin, 0.1 mu mol/L dexamethasone, 6.25 mu g/ml transferrin and 50 mu mol/L ascorbic acid phosphate;

placing the adipose-derived stem cells forming the micelles in a DMEM culture medium for culturing;

injecting the adipose-derived stem cells cultured for 7-9 days into joint cavity to transform into protochondrocytes.

Optionally, the preparation steps of the adipose-derived stem cells are as follows:

cleaning adipose tissues by using a buffer solution to remove residual blood and tissue fragments, cutting the cleaned adipose tissues into small pieces, digesting the small pieces in a shaking box for 20-60 min, standing the small pieces until the small pieces are layered, sucking upper layer adipose cell fluid, placing the adipose cell fluid in a box body at the temperature of 0-5 ℃ for culturing for 1-2 hours, sealing and centrifugally separating the box body, and removing supernatant to prepare adipose stem cell suspension;

placing the adipose-derived stem cell suspension in a cell activation medium for activation culture for 10-20 hours so as to obtain preliminarily expanded adipose-derived stem cells;

placing the adipose-derived stem cells into at least one cell culture medium and adding an activating reagent to perform adipose-derived stem cell suspension culture for 12-15 hours so as to obtain activated adipose-derived stem cells;

taking out the activated adipose-derived stem cells from the culture medium, adding normal saline, cleaning, adding enzyme solution for digestion, and culturing for three to six generations after the adipose-derived stem cells grow to 80-90% and are fused;

and naturally placing the culture solution after passage for 3-5 hours to obtain the micelle adipose-derived stem cells.

Alternatively, adipose-derived stem cells cultured for 8 days are injected into the joint cavity and transformed into protochondrocytes.

Optionally, the adipose-derived stem cells are cultured for five generations after 80% -90% of the adipose-derived stem cells are fused.

Optionally, the cell activation medium is a serum-free lymphocyte culture medium added with interleukin-2 and saperin.

Optionally, the cell activation medium is supplemented with mammalian serum.

Optionally, the mammalian serum is mammalian fetal serum.

Optionally, the activating agent is a calcium ionophore.

Optionally, the calcium ionophore is selected from a23187 or ionomycin.

The invention provides a method for activating adipose-derived stem cells to convert into protochondrocytes in vitro, aiming at activating the ASCs to convert into chondrocytes as soon as possible by fully utilizing limited time, the inventor creatively invents that the ASCs are only activated in vitro while the original activity of the ASCs is ensured, the ASCs are stimulated to an activated state, the process of converting towards a chondrocyte direction is ensured, the time point and the strength of activation are adjusted, and the ASCs in the activated state are injected into a diseased joint cavity to be continuously converted into the chondrocytes according to the original plan. This saves time and no longer emphasizes the use of three-dimensional models and a more difficult to control augmented growth environment, and the resulting chondrocytes also reduce the corresponding rejection response.

Detailed Description

It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

An in vitro method for activating the conversion of adipose stem cells into protochondrocytes, adipose tissue to be treated according to the present invention may be obtained from a patient or an immunologically acceptable donor, the adipose tissue is isolated from a fat sample, and the activated adipose tissue is cultured. An "immunologically acceptable donor" is a human having tissue, which includes adipose tissue. Firstly, washing adipose tissues by using a buffer solution to remove residual blood and tissue fragments, in the embodiment, washing by using a D-Hanks buffer solution, cutting the washed adipose tissues into small pieces, digesting in a shaking box for 20-60 min, then standing for layering, and absorbing upper-layer adipocyte liquid; placing the adipocyte liquid in a box body at the temperature of 0-5 ℃ for culturing for 1-2 hours, finding that the activity of the adipocyte liquid can be increased by utilizing the earlier stage of culturing in the box body at the temperature of 0-5 ℃ for 1-2 hours, sealing, centrifuging, removing supernatant liquid, and preparing adipose-derived stem cell suspension; placing the adipose-derived stem cell suspension in a cell activation medium for activation culture for 10-20 hours so as to obtain preliminarily expanded adipose-derived stem cells; although any cell activation medium may be used in the method of the present invention, it is preferable to culture the cells for 10 to 20 hours using a serum-free lymphocyte culture medium supplemented with interleukin-2 and saproline, so as to obtain preliminarily expanded adipose-derived stem cells; in another embodiment, the cell activation medium may further comprise mammalian serum, wherein the mammalian serum is preferably mammalian fetal serum, such as fetal bovine serum, and the amount of the mammalian serum is about 10% or 15%. The most basic nutrients for activating cells in vitro are suitable media. These media generally consist of physiological saline, amino acids, vitamins and other compounds which can be utilized directly by the cells, preferably RPMI1640 medium or serum-free medium AIM-V. The culture medium can be supplemented with mammalian immune serum, such as embryonic bovine immune serum, placing the adipose-derived stem cells in at least one cell culture medium, adding activating agent, and culturing for 12-15 hr to obtain activated adipose-derived stem cells; any cell culture medium may be used in the methods of the invention, but preferably a cell culture medium supplemented with a calcium ionophore selected from a23187 or ionomycin; in another embodiment the activating agent may also be selected from adenoviruses. Washing the activated blood cells with normal saline for at least one time, preferably washing with normal saline for two times in the embodiment of the invention, then adding enzyme liquid for digestion treatment, and culturing for three to six generations after the fat stem cells grow to 80-90% and are fused, preferably for five generations in the embodiment; naturally placing the culture solution after passage for 3-5 hours to obtain P5 generation ASCs with low immunogenicity of the micelle, and performing induction activation in vitro under the action of various stimulating factors to enable the cells to be in an activated state. Preparing a DMEM medium, wherein the proportion of the DMEM medium is 1% fetal calf serum, 10ng/ml TGF-beta, 100ng/ml BMP-6, 6.25 mu g/ml insulin, 0.1 mu mol/L dexamethasone, 6.25 mu g/ml transferrin and 50 mu mol/L ascorbic acid phosphate; placing the adipose-derived stem cells forming the micelles in a DMEM culture medium for culturing; injecting the adipose-derived stem cells cultured for 7-9 days into joint cavity to transform into protochondrocytes. Experiments prove that the relation between the culture days and the activation states of the ASCs is that the ASCs are in the optimal articular cavity injection period after being cultured for about 8 days (7-9 days), and the ASCs not only keep the original activity and homing property, but also can be accurately transformed to the original chondrocytes. Between 6 days, the ASCs were viable but had a low chance of conversion to primary chondrocytes. If more than 9 days, the activity of ASCs is reduced, and the homing property is reduced, resulting in low conversion into protochondrocytes. The optimal activation time was therefore determined to be around 8 days.

The inventor creatively invents that the ASCs are only activated in vitro while the original activity is ensured, the ASCs are stimulated to be in an activated state, the process of converting towards a cartilage cell direction is ensured, the time point and the intensity of activation are adjusted, and the ASCs in the activated state are injected into a diseased joint cavity to be continuously converted into the cartilage cell according to the original plan. This saves time without emphasizing the use of a three-dimensional model and a more difficult to control augmented growth environment, and the resulting chondrocytes also reduce the corresponding rejection response.

The incubation temperature is for example between 30 and 42 ℃. In other embodiments, between 32 and 40 ℃, or between 37 and 38 ℃, or any range contained therein. One of ordinary skill in the art will recognize that other ranges of time and temperature within these explicit ranges are contemplated and are within the present disclosure. Or any range subsumed therein. One of ordinary skill in the art will recognize that other ranges of time and temperature within these explicit ranges are contemplated and are within the present disclosure. Or any range subsumed therein. One of ordinary skill in the art will recognize that other ranges of time and temperature within these explicit ranges are contemplated and are within the present disclosure.

In the preparation of the adipose-containing stem cells related to the invention, the inventor finds that after calcium ionophore or adenovirus is added into the adipose-containing stem cells, the activated adipose-containing stem cells can stimulate the secretion amount of exosomes after entering a human body, which is 2-3 times of the secretion amount of other culture modes. The adipose-derived stem cells can be activated by contacting them with an activating agent, and the activated adipose-derived stem cells have a functional level that stimulates secretion, and the secretion amount of the exosomes is further increased by further subculturing.

Calcium ionophore is a specific metal ion species that can freely pass through lipid bilayers and soluble lipids. There are two types of ionophores: ions formed by the carrier or the channel, like adenovirus is used as the carrier, a cage-shaped structure is formed around the special ions, and the ions can freely diffuse in a hydrophobic region of a hydrophobic bilayer; channel-forming ions, such as gram-positive bacteria, form continuous liquid polar surfaces in a bilayer molecular membrane, allowing ions to diffuse through. In addition, suitable ionophores for the present invention include calcium ionophore A23187 (calcimycin), sodium salts, magnesium salts, and the like, in addition to the carriers described above. Such as calcium ionophore a23187, which are capable of responding to changes in PH gradients to concentrate calcium ions. Calcium ionophore a23187 has an acidic carboxyl group which exchanges with other cations throughout the biofilm and returns to the other end of the membrane when ion exchange is complete. The effective concentration of the ionophore is 0.05-0.5 ug/ml, and the effective concentration of the ionophore is the effective concentration for activating the adipose-derived stem cells.

While embodiments of the present invention have been described, the present invention is not limited to the above-described embodiments, which are intended to be illustrative rather than limiting, and many modifications may be made by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.