CN117467602A - Dental pulp mesenchymal stem cell microcarrier scaffold complex and preparation method and application thereof - Google Patents
Dental pulp mesenchymal stem cell microcarrier scaffold complex and preparation method and application thereof Download PDFInfo
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- CN117467602A CN117467602A CN202311407899.4A CN202311407899A CN117467602A CN 117467602 A CN117467602 A CN 117467602A CN 202311407899 A CN202311407899 A CN 202311407899A CN 117467602 A CN117467602 A CN 117467602A
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- mesenchymal stem
- dental pulp
- pulp mesenchymal
- stem cell
- microcarrier
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- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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Abstract
The application relates to the technical field of cell engineering, and particularly discloses an dental pulp mesenchymal stem cell microcarrier scaffold compound and a preparation method and application thereof. According to the application, 3D expansion is carried out under low-oxygen and high-permeability pressure conditions, and after the expansion is finished, the dental pulp mesenchymal stem cell microcarrier scaffold complex is directly subjected to stem cell liquid nitrogen freezing storage after sampling and counting. The cell scaffold complex of the dental pulp mesenchymal stem cells and the microcarrier can secrete higher cytokines, and plays a better role in anti-inflammatory and immunoregulation; the dental pulp mesenchymal stem cell microcarrier scaffold complex can be directly frozen and stored without degrading a scaffold and preparing into single cells, and has simple operation and higher activity and activity rate after cell freezing and storing and recovering; and before cell treatment, directly thawing dental pulp mesenchymal stem cell microcarrier bracket complex, adding auxiliary materials, and putting into a bioartificial liver reactor to treat acute liver failure.
Description
Technical Field
The application relates to the technical field of cell engineering, in particular to an dental pulp mesenchymal stem cell microcarrier scaffold compound and a preparation method and application thereof.
Background
Mesenchymal stem cells are derived from various tissues (such as bone marrow tissue, umbilical cord tissue, placenta tissue, adipose tissue and the like), and are a type of cells with self-replication and multi-directional differentiation capabilities. Mesenchymal stem cells can constantly self-renew and under specific conditions transform into one or more cells that constitute human tissues or organs. The mesenchymal stem cells are clinically applied to solve various refractory diseases, such as cardiovascular diseases, liver cirrhosis, nervous system diseases, repair of knee joint meniscus partial excision injury, autoimmune diseases and the like, and have great breakthrough in aspects of saving lives of more patients.
The dental pulp mesenchymal stem cells exist in dental pulp cavities, form in the cells is in a spindle shape, can be self-renewing and multi-directionally differentiated, and can be differentiated into cell line types such as fat, bone, cartilage, muscle, vascular endothelium, liver, nerve and the like through the induction of different cytokines. Dental pulp mesenchymal stem cells have stronger clonal expansion capacity and anti-inflammatory capacity than other mesenchymal stem cell types. And the dental pulp mesenchymal stem cells are abundant in source, convenient to obtain, and have natural advantages compared with other stem cell-derived tissues. Along with the development of biotechnology, dental pulp mesenchymal stem cells are increasingly researched in various disease treatment models, and have wide prospects.
Acute liver failure refers to a group of serious clinical syndromes in which massive hepatocyte necrosis and severe liver function damage occur in a short time without the original liver underlying disease, and cause hepatic encephalopathy. The clinical characteristics of the traditional Chinese medicine are that no history of chronic liver diseases exists in the past, the diseases are suddenly caused, and jaundice, liver failure, hemorrhage, neuropsychiatric symptoms and the like are rapidly caused. Multiple organ dysfunction syndrome may be combined in the short term. The disease has high death rate, belongs to one of critical diseases, and has no specific treatment method at present. Bioartificial liver is currently the most promising method of treating acute liver failure.
At present, most of cells of the bioartificial liver are liver cells, liver cancer cells and the like, and the anti-inflammatory capability is limited; liver cells are limited in source and limited in expansion capacity; liver cancer cells have little anti-inflammatory capability, and are a source of cancer cells, so that the clinical risk is high; the current cell expansion method is basically 2D adherence culture, is not convenient for the future clinical treatment and industrialization development, and has poor cell activity and biological function compared with 3D culture; the cells in the conventional 3D culture are required to be digested into single-cell cryopreservation and clinical application, and the survival rate and activity of the single-cell cryopreservation after resuscitation are greatly reduced compared with those before cryopreservation, so that the treatment effect is affected.
Disclosure of Invention
The application aims to overcome the defects of the prior art and provide a dental pulp mesenchymal stem cell microcarrier scaffold complex, a preparation method and application thereof.
In order to achieve the above purpose, the technical scheme adopted by the application is as follows:
in a first aspect, the present application provides a method for preparing an dental pulp mesenchymal stem cell microcarrier scaffold complex, comprising the steps of:
s1, inoculating dental pulp mesenchymal stem cells and microcarriers for 3D culture;
s2, introducing a suspension device containing 1% -5% of O into the 3D suspension device 2 Continuously culturing dental pulp mesenchymal stem cells and microcarriers in a serum-free culture medium of the stem cells with the osmotic pressure of 310 mM-345 mM to obtain dental pulp mesenchymal stem cell microcarrier scaffold complex, and freezing.
In the technical scheme of the application, the dental pulp mesenchymal stem cells are inoculated on microcarriers, 3D expansion is carried out under the conditions of low oxygen and high permeability, and after the expansion is finished, the cell scaffold complex of the dental pulp mesenchymal stem cells and the microcarriers is frozen after sampling and counting. The dental pulp mesenchymal stem cell microcarrier scaffold compound obtained by the application can secrete higher cytokines, and plays a better role in anti-inflammatory and immunoregulation; in addition, the dental pulp mesenchymal stem cell microcarrier scaffold complex can be directly frozen, a scaffold is not required to be degraded and single cells are prepared, the operation is simple, the activity and the activity rate of the cells after freezing and recovering are higher, and the treatment effect is improved.
According to the invention, microcarriers are used for large-scale 3D amplification, so that requirements of future clinical treatment and industrialized development are met, the operation is convenient, time and labor are saved, pollution risks are reduced, and on the other hand, the activity and biological functions of the 3D cultured cells are better than those of the 2D cultured cells.
As a preferred embodiment of the method for preparing a dental pulp mesenchymal stem cell microcarrier scaffold complex described herein, the dental pulp mesenchymal stem cells include at least one of deciduous dental pulp mesenchymal stem cells, permanent dental pulp mesenchymal stem cells and non-fully developed dental root tip stem cells. Preferably mesenchymal stem cells of deciduous teeth pulp.
The present application uses dental pulp mesenchymal stem cells, which have superior expansion capacity, anti-inflammatory capacity and immunomodulatory capacity to other stem cell types.
As a preferred embodiment of the method for preparing the dental pulp mesenchymal stem cell microcarrier scaffold complex, the passage density of the dental pulp mesenchymal stem cells is 1×10 4 Cells 5×10 4 A cell; in the culture system, the dental pulp mesenchymal stem cells are used in the following amounts: 2.5X10 4 cell/mL-5×10 4 cells/mL.
As a preferred embodiment of the preparation method of the dental pulp mesenchymal stem cell microcarrier scaffold complex, the mixed gas is N with the volume concentration of 94% 2 At a volume concentration of 5% CO 2 And is thick in volumeDegree of 1%O 2 Is a mixed gas of (a) and (b).
When the dental pulp mesenchymal stem cells and the microcarriers are amplified under the mixed gas condition of the volume concentration and the high osmotic pressure condition, higher cytokines can be secreted, and better anti-inflammatory and immunoregulatory effects are exerted. The dental pulp mesenchymal stem cell microcarrier scaffold complex can be directly frozen and stored without degrading a scaffold and preparing single cells, the operation is simple, the activity and the activity rate of the cells after freezing and recovering are higher, and the mixed gas condition of the volume concentration is favorable for maintaining the amplified saturated humidity.
As a preferred embodiment of the method for preparing dental pulp mesenchymal stem cell microcarrier scaffold complex described herein, in step S2, the stem cell serum-free medium comprises the following components in the following concentrations:
serum-free basal medium, platelet lysate with a final concentration of 4.5-5.5 wt%, vitamin C with a final concentration of 45-55 mug/ml, stem cell growth factor with a final concentration of 15-25 ng/ml, human platelet-derived growth factor with a final concentration of 15-25 ng/ml, L-glutamine with a final concentration of 1.5-2.5 mmol/ml, human transforming growth factor TGF-beta 1 with a final concentration of 90-110 ng/ml, human epidermal growth factor with a final concentration of 15-25 ng/ml, human fibroblast growth factor with a final concentration of 15-25 ng/ml;
the serum-free medium for stem cells also comprises potassium chloride with a final concentration of 1.18-1.29 wt%.
The osmotic pressure of the serum-free culture medium of the stem cells is regulated by potassium chloride with the final concentration of 1.18-1.29 wt%, so that the osmotic pressure of the serum-free culture medium of the stem cells is 310-345 mM, the osmotic pressure is improved, and the differentiation and secretion of the stem cells are further promoted.
As a preferred embodiment of the preparation method of the dental pulp mesenchymal stem cell microcarrier scaffold complex, the microcarrier comprises three-dimensional porous gelatin, wherein the porosity of the gelatin is more than 70%, the particle size is 50-500 mu m, and the uniformity is less than or equal to 100 mu m.
The microcarrier gelatin component adopted by the method is mainly convenient for digestion and degradation, the expansion efficiency is affected by too small porosity, and cells are inconvenient to inoculate; particle size too large or too small can have an effect on suspension culture and degradation; the application adopts the gelatin with the particle size, can better improve the amplification efficiency, and can not have influence on suspension culture and degradation.
In some embodiments, the temperature of the culturing is 37 ℃.
In some embodiments, the cryopreservation is a cell cryopreservation solution, which is a cryopreservation solution containing 5% dmso to 10% dmso, but is commercially available.
The dental pulp mesenchymal stem cell microcarrier scaffold complex can be stored in a higher density, the dosage of frozen stock solution is reduced, the content of DMSO entering the body is reduced, and the clinical application is safer.
In some embodiments, the cell cryopreservation density is 1×10 7 cell/mL-5×10 7 cells/mL.
In a second aspect, the present application provides an dental pulp mesenchymal stem cell microcarrier scaffold complex obtained by the above preparation method.
In a third aspect, the application provides the application of the dental pulp mesenchymal stem cell microcarrier scaffold complex in preparing a product for treating liver failure diseases.
In some embodiments, the liver failure comprises acute liver failure or chronic liver failure, preferably acute liver failure.
As a preferred embodiment of the application described herein, the dental pulp mesenchymal stem cell microcarrier scaffold complex and the auxiliary material are mixed, and the mixture is poured into a bioreactor for cell therapy.
In some embodiments, the application further comprises resuscitating the dental pulp mesenchymal stem cell microcarrier scaffold complex under water bath conditions of 37-42 ℃ for 2-4 min.
In some embodiments, the mode of treatment is biological artificial liver or combination artificial liver treatment.
As a preferred embodiment of the application described herein, the auxiliary material is a mixture of physiological saline and 5% human albumin, or the auxiliary material is a mixture of compound electrolyte and 5% human albumin, or the auxiliary material is a mixture of lactalin-format liquid and 5% human albumin.
Before cell treatment, the dental pulp mesenchymal stem cell microcarrier scaffold complex is directly thawed, auxiliary materials are added, and the dental pulp mesenchymal stem cell microcarrier scaffold complex is put into a bioartificial liver reactor to treat acute liver failure.
The culture system adopts low-oxygen and high-osmotic pressure environment, and the anti-inflammatory and immunoregulatory capacities of the produced stem cells are better than those of the common culture conditions; the dental pulp mesenchymal stem cell microcarrier scaffold compound prepared by the application can be directly frozen, does not need to be thawed into single cells for freezing, and the survival rate of the resuscitated cells after freezing is superior to that of the single cells; the recovered dental pulp mesenchymal stem cell microcarrier scaffold complex can be directly added into a bioreactor of an artificial liver system for treatment, and the operation is convenient.
Compared with the prior art, the application has the following beneficial effects:
the application provides a dental pulp mesenchymal stem cell microcarrier scaffold complex, a preparation method and application thereof. The cell scaffold complex of the dental pulp mesenchymal stem cells and the microcarrier prepared by the application can secrete higher cytokines, and has better anti-inflammatory and immunoregulatory effects; the dental pulp mesenchymal stem cell microcarrier scaffold complex can be directly frozen and stored without degrading a scaffold and preparing into single cells, and has simple operation and higher activity and activity rate after cell freezing and storing and recovering; in addition, the dental pulp mesenchymal stem cell microcarrier scaffold complex can be stored in a higher density, the dosage of the frozen stock solution is reduced, the content of DMSO entering the body is reduced, and the clinical application is safer. Before cell treatment, directly thawing dental pulp mesenchymal stem cell microcarrier bracket complex, adding auxiliary materials, and putting into a bioartificial liver reactor to treat acute liver failure.
Drawings
FIG. 1 is a cell phenotype diagram of dental pulp mesenchymal stem cells;
FIG. 2 is a graph showing the comparison of 3D culture expansion curves and 2D culture expansion curves of dental pulp mesenchymal stem cells under the same low-oxygen high-osmotic pressure condition;
FIG. 3 shows the inhibitory effect of dental pulp mesenchymal stem cells on secreted TNF-alpha cytokines under 3D low oxygen high osmotic pressure culture conditions, 3D low oxygen normal osmotic pressure culture conditions, 3D normal oxygen normal osmotic pressure culture conditions, 2D low oxygen high osmotic pressure culture conditions, 2D normal oxygen normal osmotic pressure culture conditions, and co-incubation conditions with 5-fold amount of PBMC cells.
Fig. 4 is a resuscitatory staining photograph of a dental pulp mesenchymal stem cell microcarrier scaffold complex after cryopreservation, wherein green represents living cells, high-brightness red represents dead cells, and dark red is a background color of microcarrier staining.
Fig. 5 is a comparison of the viability of dental pulp mesenchymal stem cells after single cell cryopreservation and resuscitation compared to the viability of dental pulp mesenchymal stem cell microcarrier scaffold complex after cryopreservation.
Fig. 6 is a schematic diagram of application of dental pulp mesenchymal stem cell microcarrier scaffold complex in treating acute liver failure mini-pigs by artificial liver.
FIG. 7 is a schematic representation of survival of mini-pigs in acute liver failure treated with bioartificial liver using dental pulp mesenchymal stem cell microcarrier scaffold complexes.
FIG. 8 is a schematic diagram showing the detection of serum biochemical indexes related to liver injury, such as blood ammonia content (FIG. 8-A) and glutamic pyruvic transaminase content (FIG. 8-B) of a miniature pig with acute liver failure by using a biological artificial liver of dental pulp mesenchymal stem cell microcarrier scaffold complex.
FIG. 9 is a schematic representation of liver inflammatory response-related gene expression in mini-pigs with acute liver failure using bioartificial livers of dental pulp mesenchymal stem cell microcarrier scaffold complexes.
Detailed Description
In order that the invention may be readily understood, a more complete description of the invention will be rendered by reference to the appended drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
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. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available.
Example 1, dental pulp mesenchymal stem cell microcarrier scaffold complex and preparation method thereof
The embodiment 1 provides a preparation method of an dental pulp mesenchymal stem cell microcarrier scaffold complex, which comprises the following steps:
the dental pulp mesenchymal stem cells (refer to FIG. 1) with the excessive cell phenotype are identified by a flow cytometer according to the 2.5X10 6 The number of individual living cells was inoculated into 125mL 3D suspension culture flasks, 50mL of the serum-free medium for stem cells was added to the flasks, and the osmotic pressure of the serum-free medium for stem cells was measured to be 328mM. Then 100mg of microcarrier sterilized by irradiation is added into a rotary bottle, the rotating speed is set to be 45rpm, and the volume concentration of N is 94 percent 2 At a volume concentration of 5% CO 2 And a volume concentration of 1%O 2 Under the mixed gas condition (hypoxia condition), the culture is continuously carried out for 48 hours in 3D, and the culture temperature is 37 ℃. And then supplementing 25mL of stem cell serum-free culture medium into the rotary bottle, regulating the rotating speed to 60rpm, and continuing to perform hypoxia culture for 48 hours to obtain the dental pulp mesenchymal stem cell microcarrier scaffold complex.
Transferring the suspension of the dental pulp mesenchymal stem cell microcarrier scaffold complex after amplification into a 500mL centrifuge bottle, and joltAfter mixing, 1mL of the mixture is sampled, and after standing for 1min, the supernatant is removed; 200 microliters of 3D was then added to the dental pulp mesenchymal stem cell microcarrier scaffold complexThe Digest lysate was placed in a 37℃water bath for 30min to lyse the microcarriers, during which time the microcarriers were accelerated by pipetting at 5-10 intervals of 10 min. After the dental pulp mesenchymal stem cell microcarrier scaffold complex is completely cracked, calculating the activity rate and the cell number of dental pulp mesenchymal stem cells in 1mL of suspension by using an NC-200 counter, and calculating the cell number and the activity rate obtained after amplification. As shown in FIG. 2, the 3D culture expansion curve and the 2D culture expansion curve of dental pulp mesenchymal stem cells are compared, and the dental pulp mesenchymal stem cell expansion efficiency of the 3D culture system is slightly higher than that of the 2D culture system. .
Centrifuging 180g of 500mL centrifuge bottle loaded with suspension of dental pulp mesenchymal stem cell microcarrier scaffold complex for 5min, removing supernatant, and mixing according to 2×10 7 Adding the CS10 frozen stock solution with calculated volume into the ratio of living cells/mL frozen stock solution, blowing uniformly by a pipettor, adding into a frozen stock wheat tube or a frozen stock bag, then placing into a programmed cooling instrument for programmed cooling, and transferring into a liquid nitrogen tank for long-term freezing storage after cooling is finished.
The serum-free medium for stem cells comprises the following components in concentration:
serum-free basal medium, platelet lysate at a final concentration of 5.0wt%, vitamin C at a final concentration of 50 μg/ml, stem cell growth factor at a final concentration of 20ng/ml, human platelet-derived growth factor at a final concentration of 20ng/ml, L-glutamine at a final concentration of 2.0mmol/ml, human transforming growth factor TGF- β1 at a final concentration of 100ng/ml, human epidermal growth factor at a final concentration of 20ng/ml, human fibroblast growth factor at a final concentration of 20 ng/ml.
The stem cell serum-free medium also included potassium chloride at a final concentration of 1.24 wt%.
As shown in fig. 1, the dental pulp mesenchymal stem cells highly express mesenchymal stem cell markers such as CD73, CD90, CD105 and the like, and the positive proportion is more than 95%; the markers of CD11b, CD19, CD45, CD34, HLA-DR and the like are expressed in low proportion, the proportion of the markers is lower than 2%, and the identification requirements of the industry on mesenchymal stem cells are met.
Example 2
Compared with example 1, the osmotic pressure of the serum-free medium of the stem cells is 310mM, and the specific formula of the serum-free medium of the cells is as follows: platelet lysate at a final concentration of 5.0wt%, vitamin C at a final concentration of 50. Mu.g/ml, stem cell growth factor at a final concentration of 20ng/ml, human platelet-derived growth factor at a final concentration of 20ng/ml, L-glutamine at a final concentration of 2.0mmol/ml, human transforming growth factor TGF-beta 1 at a final concentration of 100ng/ml, human epidermal growth factor at a final concentration of 20ng/ml, human fibroblast growth factor at a final concentration of 20ng/ml, potassium chloride at a final concentration of 1.18 wt%.
The rest steps, parameters and reagents are the same as in example 1, and dental pulp mesenchymal stem cell microcarrier scaffold complex is obtained.
Example 3
Compared with example 1, the osmotic pressure of the serum-free medium of the stem cells is 345mM, and the specific formula of the serum-free medium of the cells is as follows: platelet lysate at a final concentration of 5.0wt%, vitamin C at a final concentration of 50. Mu.g/ml, stem cell growth factor at a final concentration of 20ng/ml, human platelet-derived growth factor at a final concentration of 20ng/ml, L-glutamine at a final concentration of 2.0mmol/ml, human transforming growth factor TGF-beta 1 at a final concentration of 100ng/ml, human epidermal growth factor at a final concentration of 20ng/ml, human fibroblast growth factor at a final concentration of 20ng/ml, potassium chloride at a final concentration of 1.29 wt%.
The rest steps, parameters and reagents are the same as in example 1, and dental pulp mesenchymal stem cell microcarrier scaffold complex is obtained.
Comparative example 1
In comparison to example 1, 3D normoxic normovolemic culture conditions were used:
the incubator environment is air with volume concentration of 95% and CO with volume concentration of 5% 2 3D culture was performed under the mixed gas conditions (normoxic conditions).
The serum-free culture medium comprises 5.0wt% of platelet lysate, 50 mug/ml of vitamin C, 20ng/ml of stem cell growth factor, 20ng/ml of human platelet-derived growth factor, 2.0mmol/ml of L-glutamine, 100ng/ml of human transforming growth factor TGF-beta 1, 20ng/ml of human epidermal growth factor and 20ng/ml of human fibroblast growth factor. The osmotic pressure of the serum-free medium of the stem cells was measured to be 290mM.
The rest steps, parameters and reagents are the same as in example 1, and dental pulp mesenchymal stem cell microcarrier scaffold complex is obtained.
Comparative example 2
In comparison to example 1, 2D low oxygen high osmotic pressure culture conditions were used:
2.5X10 of the excess cell phenotype will be identified by flow cytometry 6 Single cells of dental pulp mesenchymal stem cells according to 1.1X10 4 Individual cells/cm 2 The medium was inoculated in a T225 flask for expansion culture, 75mL of the serum-free medium was added to the flask, and the osmotic pressure of the serum-free medium was measured to be 328mM. At a volume concentration of 94% N 2 At a volume concentration of 5% CO 2 And a volume concentration of 1%O 2 Under the mixed gas condition (hypoxia condition), the culture is continuously carried out for 96 hours in 2D, and the culture temperature is 37 ℃. Then, 25mL of serum-free medium for stem cells was supplemented into the flask, the hypoxia culture was continued for 48 hours, cell digestion was performed using trypsin substitute Tryple digest, and after centrifugation to remove the supernatant, 2 times of washing was performed using DPBS solution to obtain 2D-cultured dental pulp mesenchymal stem cells.
The serum-free medium for stem cells comprises the following components in concentration:
serum-free medium, platelet lysate at a final concentration of 5.0wt%, vitamin C at a final concentration of 50 μg/ml, stem cell growth factor at a final concentration of 20ng/ml, human platelet-derived growth factor at a final concentration of 20ng/ml, L-glutamine at a final concentration of 2.0mmol/ml, human transforming growth factor TGF- β1 at a final concentration of 100ng/ml, human epidermal growth factor at a final concentration of 20ng/ml, human fibroblast growth factor at a final concentration of 20ng/ml, potassium chloride at a final concentration of 1.24 wt%.
Comparative example 3
In comparison to example 1, 2D normoxic normovolemic culture conditions were used:
2.5X10 of the excess cell phenotype will be identified by flow cytometry 6 Single cells of dental pulp mesenchymal stem cells according to 1.1X10 4 Individual cells/cm 2 The medium was inoculated in a T225 flask for expansion culture, 75mL of the serum-free medium was added to the flask, and the osmotic pressure of the serum-free medium was measured to be 290mM. At a volume concentration of 95% air and a volume concentration of 5% CO 2 Under the mixed gas condition (normal oxygen condition), the culture is continuously carried out for 96 hours in 2D, and the culture temperature is 37 ℃. Then, 25mL of serum-free medium for stem cells was supplemented to the flask, and the normal oxygen culture was continued for 48 hours, and cell digestion was performed using a trypsin substitute Tryple digest, and after the supernatant was removed by centrifugation, 2 times of washing was performed using a DPBS solution to obtain 2D-cultured dental pulp mesenchymal stem cells.
The serum-free medium for stem cells comprises the following components in concentration:
serum-free medium, platelet lysate at a final concentration of 5.0wt%, vitamin C at a final concentration of 50 μg/ml, stem cell growth factor at a final concentration of 20ng/ml, human platelet-derived growth factor at a final concentration of 20ng/ml, L-glutamine at a final concentration of 2.0mmol/ml, human transforming growth factor TGF- β1 at a final concentration of 100ng/ml, human epidermal growth factor at a final concentration of 20ng/ml, human fibroblast growth factor at a final concentration of 20 ng/ml.
Comparative example 4
In comparison to example 1, 3D hypoxic normal osmotic pressure culture conditions were used:
the incubator environment was 94% N by volume 2 At a volume concentration of 5% CO 2 And a volume concentration of 1%O 2 3D culture was performed under the mixed gas conditions (hypoxia conditions).
The stem cell serum-free medium comprises the following components in concentration:
serum-free medium, platelet lysate at a final concentration of 5.0wt%, vitamin C at a final concentration of 50 μg/ml, stem cell growth factor at a final concentration of 20ng/ml, human platelet-derived growth factor at a final concentration of 20ng/ml, L-glutamine at a final concentration of 2.0mmol/ml, human transforming growth factor TGF- β1 at a final concentration of 100ng/ml, human epidermal growth factor at a final concentration of 20ng/ml, human fibroblast growth factor at a final concentration of 20 ng/ml. The osmotic pressure of the serum-free medium of the stem cells was measured to be 290mM.
The rest steps, parameters and reagents are the same as in example 1, and dental pulp mesenchymal stem cell microcarrier scaffold complex is obtained.
Comparative example 5
In comparison with example 1, 3D normoxic high osmotic pressure culture conditions were used:
the incubator environment is air with volume concentration of 95% and CO with volume concentration of 5% 2 3D culture was performed under the mixed gas conditions (normoxic conditions).
The stem cell serum-free medium comprises the following components in concentration:
serum-free medium, platelet lysate at a final concentration of 5.0wt%, vitamin C at a final concentration of 50 μg/ml, stem cell growth factor at a final concentration of 20ng/ml, human platelet-derived growth factor at a final concentration of 20ng/ml, L-glutamine at a final concentration of 2.0mmol/ml, human transforming growth factor TGF- β1 at a final concentration of 100ng/ml, human epidermal growth factor at a final concentration of 20ng/ml, human fibroblast growth factor at a final concentration of 20ng/ml, potassium chloride at a final concentration of 1.24 wt%. The osmotic pressure of the serum-free medium of the stem cells was measured to be 328mM.
The rest steps, parameters and reagents are the same as in example 1, and dental pulp mesenchymal stem cell microcarrier scaffold complex is obtained.
The dental pulp mesenchymal stem cells obtained in example 1 and comparative examples 1 to 5 were incubated with 5-fold amount of PBMC cells, and the inhibitory effect on the secreted TNF-. Alpha.cytokines was compared.
As shown in FIG. 3, the inhibitory effect of dental pulp mesenchymal stem cells on secreted TNF-alpha cytokines under the condition of 3D hypoxia and high osmotic pressure is the highest, which indicates that the dental pulp mesenchymal stem cells cultured under the condition have the best anti-inflammatory effect. Meanwhile, under the same conditions, the inhibition effect of the 3D cultured dental pulp mesenchymal stem cells on the secreted TNF-alpha cytokines is higher than that of the 2D cultured dental pulp mesenchymal stem cells; the inhibition effect of the dental pulp mesenchymal stem cells cultured under the condition of low oxygen and high osmotic pressure on the secreted TNF-alpha cytokines is higher than that of the dental pulp mesenchymal stem cells cultured under the condition of normal oxygen and normal osmotic pressure.
Test example and application of dental pulp mesenchymal stem cell microcarrier scaffold complex in treating acute liver failure in artificial liver support system
The total cell number was about 1X 10 9 Taking out the freezing tube or freezing bag of individual cells from a liquid nitrogen tank, thawing in a water bath at 37 ℃ for 3min, transferring all suspension of dental pulp mesenchymal stem cell microcarrier scaffold complex (prepared in example 1) in the freezing tube or freezing bag into a 500mL centrifugal bottle, mixing uniformly in a reverse manner, sampling 2mL of the suspension into two tubes of 1mL each, standing one tube for 1min, removing the supernatant, adding 100 mu L of live and dead fluorescent dye solution into the cell microcarrier complex, incubating for 15-30 min in a dark place, removing the supernatant, washing for 1 time by using DPBS, photographing by using a fluorescent microscope, and observing the state of the cells. After another tube was left to stand for 1min, the supernatant was removed, and then 200. Mu.l of 3D was added to the dental pulp mesenchymal stem cell microcarrier scaffold complexThe Digest lysate was placed in a 37℃water bath for 30min to lyse the microcarriers, during which time the microcarriers were accelerated by pipetting at 5-10 intervals of 10 min. After the microcarrier is completely cracked, the viability and the cell number of dental pulp mesenchymal stem cells in 1mL of suspension are calculated by an NC-200 counter, so that the cell number and the viability after resuscitation are calculated.
180g of a 500mL centrifuge bottle loaded with a suspension of dental pulp mesenchymal stem cell microcarrier scaffold complex is centrifuged for 5min, supernatant is removed, the mixture is washed with physiological saline for 2 times, 1L of physiological saline plus 5% human albumin is added to prepare a final preparation, the preparation is filled into a sterile bioreactor, the sterile bioreactor is filled into a bioartificial liver support system, and the bioartificial liver treatment is carried out on a miniature pig with liver failure in vitro for 6h.
In the application, 12 miniature pigs (about 30kg in weight) are selected to establish an acute liver injury model, 0.4g/kg D-Gal is injected into the miniature pigs on the 0 th day to induce acute liver failure, and 24h experimental group miniature pigs are subjected to bioartificial liver treatment after induction. 6 small pigs are randomly selected as experimental groups to treat a bioartificial liver system loaded with dental pulp mesenchymal stem cell microcarrier complex biological agents; 6 were used as model controls without any treatment. The survival of the minipigs was recorded by continuous observation for 14 days after the end of the treatment. The method comprises the steps of taking blood from the piglets every day before molding, detecting various functional indexes of liver failure, and stopping sample collection from the 7 th day after treatment or from the death of the piglets. And simultaneously, the inflammatory response related genes of the liver of the miniature pig are detected. Liver tissues of small pigs (day 14) of the bioartificial liver treatment group and small pigs of the acute liver failure model control group were randomly sampled and tissue mRNA was extracted for QPCR detection, respectively.
Fig. 4 is a resuscitatory staining photograph of a dental pulp mesenchymal stem cell microcarrier scaffold complex after cryopreservation, wherein green represents living cells, high-brightness red represents dead cells, and dark red is a background color of microcarrier staining. When the dental pulp mesenchymal stem cell microcarrier scaffold compound is revived and stained after freezing, cells are basically full of colors, almost no red color exists, and the dental pulp mesenchymal stem cells in the compound are almost living cells after freezing and reviving.
The comparison of the survival rate of the dental pulp mesenchymal stem cells before and after the freezing and the survival rate of the dental pulp mesenchymal stem cells microcarrier scaffold complex (i.e. the cell microcarrier complex) before and after the freezing and the recovery is shown in fig. 5. As shown in fig. 5, after 3 months of freezing, the survival rate of single cells is greatly reduced, while the survival rate of the dental pulp mesenchymal stem cell microcarrier scaffold complex is less reduced after the resuscitation, which indicates that the dental pulp mesenchymal stem cell microcarrier scaffold complex can effectively maintain the cell state in the complex in the freezing process, and effectively avoid the reduction of the cell survival rate in the freezing process.
A schematic of the application of the dental pulp mesenchymal stem cell microcarrier scaffold complex in treating small pigs with acute liver failure by artificial liver is shown in FIG. 6. As shown in fig. 7, 83% of the experimental group minipigs survived for more than 7 days, while untreated minipigs all died within 4 days; as shown in fig. 8, the detection results of the liver-related serum biochemical indexes show that the liver failure-related indexes are obviously up-regulated in the first day of the induction of failure for both groups of miniature pigs, and the miniature pig indexes of the treatment group gradually drop after the biological artificial liver treatment until reaching the level consistent with the origin on the 7 th day; the index of the miniature pigs in the control group is continuously increased or stabilized at a higher level; as shown in FIG. 9, the control group of piglets was significantly higher than the treatment group of piglets with respect to the expression of the inflammatory response-related gene. Therefore, the dental pulp mesenchymal stem cell microcarrier bracket compound can effectively play roles in clearing toxin and regulating inflammation by stem cells in an artificial liver support system, and further promote self-repair of liver functions so as to play a role in treating liver failure.
Finally, it should be noted that the above embodiments are only for illustrating the technical solutions of the present application and not for limiting the scope of protection of the present application, and although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solutions of the present application without departing from the spirit and scope of the technical solutions of the present application.
Claims (10)
1. A preparation method of an dental pulp mesenchymal stem cell microcarrier scaffold complex, which is characterized by comprising the following steps:
s1, inoculating dental pulp mesenchymal stem cells and microcarriers for 3D culture;
s2, introducing a suspension device containing 1% -5% of O into the 3D suspension device 2 Continuously culturing dental pulp mesenchymal stem cells and microcarriers in a serum-free culture medium of the stem cells with the osmotic pressure of 310 mM-345 mM to obtain dental pulp mesenchymal stem cell microcarrier scaffold complex, and freezing.
2. The method of preparing a dental pulp mesenchymal stem cell microcarrier scaffold complex according to claim 1, wherein the dental pulp mesenchymal stem cells comprise at least one of deciduous dental pulp mesenchymal stem cells, permanent dental pulp mesenchymal stem cells and non-fully developed dental root tip stem cells.
3. The method for preparing dental pulp mesenchymal stem cell microcarrier scaffold complex according to claim 1, wherein the passage density of dental pulp mesenchymal stem cells is 1 x 10 4 Cells 5×10 4 A cell; in the culture system, the dental pulp mesenchymal stem cells are used in the following amounts: 2.5X10 4 cell/mL-5×10 4 cells/mL.
4. The method for preparing dental pulp mesenchymal stem cell microcarrier scaffold complex according to claim 1, wherein the mixed gas is 94% N by volume 2 At a volume concentration of 5% CO 2 And a volume concentration of 1%O 2 Is a mixed gas of (a) and (b).
5. The method of preparing dental pulp mesenchymal stem cell microcarrier scaffold complex according to claim 1, wherein in step S2, the stem cell serum-free medium comprises the following components in the following concentrations:
serum-free basal medium, platelet lysate with a final concentration of 4.5-5.5 wt%, vitamin C with a final concentration of 45-55 mug/ml, stem cell growth factor with a final concentration of 15-25 ng/ml, human platelet-derived growth factor with a final concentration of 15-25 ng/ml, L-glutamine with a final concentration of 1.5-2.5 mmol/ml, human transforming growth factor TGF-beta 1 with a final concentration of 90-110 ng/ml, human epidermal growth factor with a final concentration of 15-25 ng/ml, human fibroblast growth factor with a final concentration of 15-25 ng/ml;
the serum-free medium for stem cells also comprises potassium chloride with a final concentration of 1.18-1.29 wt%.
6. The method for preparing dental pulp mesenchymal stem cell microcarrier scaffold complex according to claim 5, wherein the microcarrier comprises three-dimensional porous gelatin, the porosity of the gelatin is more than 70%, the particle size is 50-500 μm, and the uniformity is less than or equal to 100 μm.
7. Dental pulp mesenchymal stem cell microcarrier scaffold complex obtained by the preparation method according to any one of claims 1-6.
8. Use of a dental pulp mesenchymal stem cell microcarrier scaffold complex according to claim 7 for the preparation of a product for the treatment of liver failure disease.
9. The use according to claim 8, wherein the dental pulp mesenchymal stem cell microcarrier scaffold complex and the auxiliary material are mixed, and the mixture is poured into a bioreactor for cell therapy.
10. The use according to claim 9, wherein the auxiliary material is a mixture of physiological saline and 5% human albumin, or wherein the auxiliary material is a mixture of a compound electrolyte and 5% human albumin, or wherein the auxiliary material is a mixture of lactate in-line solution and 5% human albumin.
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