CN112662618A - Umbilical cord mesenchymal stem cells and preparation method thereof - Google Patents
Umbilical cord mesenchymal stem cells and preparation method thereof Download PDFInfo
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Abstract
The invention relates to umbilical cord mesenchymal stem cells and a preparation method thereof. The preparation method comprises the following steps: (1) perforating the sheet umbilical cord tissue; (2) placing the cell apoptosis product in the hole formed in the step (1) for pre-culture; (3) removing the cell tar-death product in the hole, and culturing the obtained umbilical cord tissue; the cell tar death product comprises at least one of cell tar death secretion, cell tar death extract and tar death cells generated after cell tar death. The method can obtain a large amount of umbilical cord mesenchymal stem cells with excellent properties in a short time, and has the advantages of simple operation process, stable experimental result and no obvious difference between the properties of the cells obtained by each batch of culture.
Description
Technical Field
The invention relates to the field of stem cell culture, in particular to umbilical cord mesenchymal stem cells and a preparation method thereof.
Background
A stem cell is a cell having self-replication ability and differentiation potential, and can be differentiated into various functional cells under certain conditions. The differentiation potential can be classified into totipotent stem cells, pluripotent stem cells and multipotent stem cells. Although the pluripotent stem cells do not have the potential of forming complete donors, the pluripotent stem cells have the differentiation potential of differentiating into different germ layer cells and are not limited by ethics, particularly mesenchymal stem cells belonging to the pluripotent stem cells are discovered to have low immunogenicity, paracrine characteristics and immunoregulation effect, are paid more and more attention by researchers, and show bright application prospects in the field of clinical treatment. Mesenchymal stem cells are widely distributed in bone marrow, fat, umbilical cord blood and umbilical cord. The umbilical cord mesenchymal stem cells become an ideal source for research of the mesenchymal stem cells due to wide sources, convenient collection and noninvasive material collection of the umbilical cord mesenchymal stem cells.
Umbilical cord mesenchymal stem cells can be isolated from the epithelial layer of umbilical cord tissue, wharton's jelly, perivascular tissue and umbilical cord blood, wherein wharton's jelly, perivascular tissue and umbilical cord blood are the main sources of umbilical cord mesenchymal stem cells. In addition, the umbilical cord mesenchymal stem cells have the following characteristics: 1. has strong proliferation capacity and multidirectional differentiation potential. The umbilical cord mesenchymal stem cells still have good dryness after being subjected to multiple passages, and can be differentiated into cells of three embryonic sources under the condition of applying proper induction conditions under the condition of being suitable for in vivo environment or in vitro culture. For example, umbilical cord mesenchymal stem cells can differentiate into endodermal-derived hepatocytes, mesodermal-derived cardiomyocytes, and ectodermal-derived neural cells. 2. The immunogenicity is low, the umbilical cord mesenchymal stem cells do not express MHC-II and costimulatory molecules CD80 and CD86, and the level of MHC-I molecules is low, so that the umbilical cord mesenchymal stem cells show extremely low immunogenicity. 3. Has the function of immunoregulation, and umbilical cord mesenchymal stem cells positively express immunosuppressive molecules HLA-G, indoleamine 2, 3-dioxygenase (IDO), prostaglandin E2(PGE2), interleukin-6 (IL-6) and interleukin-10 (IL-10), thereby inhibiting over-secretion of TNF-alpha of monocytes and over-proliferation of T lymphocytes, realizing the alleviation of inflammatory reaction and the inhibition of over-immune reaction of organisms. 4. The umbilical cord mesenchymal stem cells can release growth factors such as insulin-like growth factors (IGF), Vascular Endothelial Growth Factors (VEGF), Hepatocyte Growth Factors (HGF) and transforming growth factor-beta (TGF-beta) into the environment through paracrine action, and promote the growth of cells and the regeneration of tissues. The characteristics show that the umbilical cord mesenchymal stem cells have great treatment potential in the fields of autoimmune disease, tissue repair and the like
Although the umbilical cord mesenchymal stem cells have good application prospect, the standard umbilical cord mesenchymal stem cells do not exist at the present stageThe primary culture mode of the mesenchymal stem cells can stably obtain the primary cells with good properties. The existing primary culture method of umbilical cord mesenchymal stem cells is mainly divided into a wall attaching method and an enzyme digestion method. The wall pasting method can be divided into a tissue block direct wall pasting method, a tissue homogenate wall pasting method and a digested tissue wall pasting method. The enzymatic digestion method can be further divided into a complete digestion method and a stepwise digestion method. The method for obtaining the umbilical cord mesenchymal stem cells by the wall pasting method mainly comprises the steps of cleaning and separating umbilical cord tissues to obtain Wharton jelly in the umbilical cord, and then dividing the Wharton jelly tissues into 1mm in size3～2mm3The small tissue pieces are placed in a culture dish or a culture flask for primary culture of cells. The tissue homogenate wall pasting method is operated by further crushing the obtained Wharton jelly tissue to obtain minced meat-like tissue fragments, and then attaching the minced tissue fragments to a culture dish or a culture bottle for primary culture. Compared with a tissue homogenate method adopting a direct adherence method, the tissue homogenate method has the advantages of smaller tissue blocks, larger specific surface area and more contribution to emigration of cells. The complete digestion method is to digest the obtained wharton jelly tissue by using biological enzyme until the tissue completely disappears, so as to obtain a large amount of umbilical cord mesenchymal stem cells. Distributed digestion is also performed by digesting wharton's jelly tissue with biological enzymes, but during digestion, digestion is temporarily suspended, and the digested cells are harvested by centrifugation of the digestive fluid, followed by digestion of the tissue, and the digestion step is repeated until the tissue mass is completely digested. Compared with a complete digestion method, the distributed digestion method collects cells digested first in advance, and reduces damage of digestive enzymes to the cells. The adherence method has a better cell state than the umbilical cord mesenchymal stem cells obtained by a digestion method, but the adherence method has a longer primary culture period (14-21day) and cannot obtain a large amount of umbilical cord mesenchymal stem cells in a short time. Digestion can obtain a large number of cells in a short period of time, but damage to the cells by biological enzymes cannot be avoided. At present, a method combining a wall pasting method and a digestion method, namely a tissue wall pasting method after digestion, is adopted, and the specific operation method is to divide the obtained Wharton jelly tissue into 8mm3～64mm3After the tissue blocks with the size, the tissue blocks are digested by pancreatin with the concentration of 1.1-1.4 g/L for 10min-30 min, digesting 0.06 wt% -0.09 wt% collagenase for 40 min-80 min; the digested tissue pieces were washed and cultured. Although treatment of tissue with biological enzymes increases the speed of cell migration, cell migration is still slow, primary cell culture cycles are still long, and the activity of each cell obtained cannot be determined.
In the existing umbilical cord mesenchymal stem cell primary culture mode, the wall pasting method is to climb out from a tissue block through the migration capacity of cells, or to increase the surface area through reducing the volume of the tissue block so as to promote the cells to rapidly migrate out from the tissue. The digestion method is to directly obtain a large number of cells by utilizing the digestion effect of biological enzymes and acting on rigid structures in the umbilical cord tissue to release the cells from the umbilical cord tissue. The principle of promoting cells to migrate quickly by the tissue adherence method after digestion is similar to that of the digestion method, the digestion effect of biological enzyme is utilized to act on a rigid skeleton structure in the umbilical cord tissue, the difference lies in that the digestion method completely degrades the skeleton structure to directly obtain the cells, the tissue adherence method after digestion avoids the damage of the biological enzyme to the cells, the skeleton structure in the umbilical cord tissue is not completely digested, the biological enzyme is utilized to loosen the tissue skeleton, the space gap between the skeleton structures is increased, and the cells are more favorable for migrating out of the umbilical cord tissue. Although the above primary culture methods can obtain umbilical cord mesenchymal stem cells, they all have disadvantages. The digestion method cannot avoid the damage of biological enzyme to cells, and although a large amount of cells are obtained, a large amount of hybrid cells of non-umbilical cord mesenchymal stem cells are mixed in the digestion method. The primary culture period of the adherence method is too long, a large amount of umbilical cord mesenchymal stem cells cannot be rapidly obtained, although the tissue adherence method after digestion can effectively accelerate the emigration of the primary cells, the primary culture period is limited, and the umbilical cord mesenchymal stem cells of the primary culture each time cannot be guaranteed to have good biological activity. Therefore, a cell culture technique capable of rapidly and stably obtaining a large number of primary stem cells with good properties is important.
Disclosure of Invention
Based on this, the present invention aims to provide a method for rapidly obtaining umbilical cord mesenchymal stem cells with stronger biological activity in large quantities.
The specific technical scheme is as follows:
a preparation method of umbilical cord mesenchymal stem cells comprises the following steps:
(1) perforating the sheet umbilical cord tissue;
(2) placing the cell apoptosis product in the hole formed in the step (1) for pre-culture;
(3) removing the cell tar-death product in the hole, and culturing the obtained umbilical cord tissue;
the cell tar death product comprises at least one of cell tar death secretion, cell tar death extract and tar death cells generated after cell tar death.
In some of these embodiments, the area of the perforations comprises 10% to 90% of the area of the entire sheet of umbilical cord tissue.
In some of these embodiments, the holes have a diameter of 0.1cm to 10cm, with a preferred diameter range of 0.5 to 3 cm.
In some of these embodiments, the distribution density of the pores on the umbilical cord tissue is 25cm per umbilical cord tissue210-60 holes are punched.
In some of these embodiments, the aperture is a through aperture.
In some of these embodiments, the cell apoptosis product of step (2) is mixed with a gel matrix and the resulting mixture is placed into the pores.
In some of these embodiments, the gel matrix is at least one of a native polypeptide hydrogel and a native biogel. Preferably, the native biogel is at least one of fibrin gel, extracellular matrix gel and collagen gel.
In some embodiments, the protein concentration in the mixture of the cell apoptosis product of step (2) and the gel matrix is 1ng/ml to 10mg/ml, preferably 20ng/ml to 1 mg/ml.
In some embodiments, the pre-culturing time in step (2) is 1-148 h, preferably 6-48 h.
In some embodiments, the cells of step (2) comprise at least one of monocytes, fibroblasts, endothelial cells, smooth muscle cells, neural cells, cardiac muscle cells, corneal stromal cells, totipotent stem cells, pluripotent stem cells, and unipotent stem cells. Bone marrow mesenchymal stem cells, limbal stem cells or cardiomyocyte-derived cells may be mentioned as examples.
In some of these embodiments, the method of inducing a cell to produce the cellular apoptosis product comprises: physical induction, chemical induction or biological induction.
In some embodiments, the physical induction method comprises any one or more of ultraviolet irradiation induction, ultrasonic induction, radiation stimulation induction, and hydroxypropyl cellulose caprylate coated dish culture.
In some embodiments, the chemical induction method comprises any one or more of perforin induction, urate crystallization induction and flagellin induction.
In some of these embodiments, the biological induction method comprises inducing infection of the cell with any one or more microorganisms selected from the group consisting of pseudomonas, listeria, shigella, legionella, pseudomonas aeruginosa, francisella, yersinia, streptococcus pneumoniae, actinobacillus pleuropneumoniae, candida albicans, staphylococcus aureus, salmonella typhi, hepatitis virus, and immunodeficiency virus.
In some of these embodiments, the sheet of umbilical cord tissue comprises post-skinned, deployed umbilical cord tissue with or without arteries or veins.
In some of these embodiments, the means for pre-culturing comprises a fully submerged culture and a partially submerged culture.
In some of these embodiments, the medium of step (2) the pre-culturing and (3) the continuing culturing is complete medium.
In some of these embodiments, the source of the umbilical cord tissue is a human or non-human mammal.
In some of these embodiments, the non-human mammal comprises a dog, pig, or horse.
The invention also aims to provide the umbilical cord mesenchymal stem cells prepared by the preparation method.
Compared with the prior art, the invention has the following beneficial effects:
the invention realizes a method for rapidly obtaining umbilical cord mesenchymal stem cells with stronger biological activity in large quantities for the first time. The method comprises the steps of punching a sheet umbilical cord tissue for the first time, placing a cell apoptosis product in the hole for culturing, constructing a local inflammatory environment through the cell apoptosis product, pre-stimulating the umbilical cord tissue and using the pre-stimulated umbilical cord tissue as a driving force for migration of umbilical cord mesenchymal stem cells to promote the rapid migration of the activated umbilical cord mesenchymal stem cells, selectively retaining the umbilical cord mesenchymal stem cells with good growth state, and finally and rapidly obtaining a large amount of primary umbilical cord mesenchymal stem cells with stronger migration, proliferation, paracrine and immunosuppressive activities.
The method avoids excessive mechanical force action and biological enzyme digestion action in the prior method, and ensures the activity of stem cells in umbilical cord tissues.
In addition, the method is simple to operate and strong in repeatability, and the cell character obtained each time is stable.
Drawings
FIG. 1 is a schematic diagram of the construction of a localized inflammatory environment within perforated, sheet-like umbilical cord tissue using a gel containing inflammatory factors of cellular apoptosis;
FIG. 2 is a photograph of primary cultured umbilical cord mesenchymal stem cells taken with a light microscope.
FIG. 3 is a photo-scope photograph of focal-dead bodies formed by culturing bone marrow mesenchymal stem cells (BMSCs) on OPC dishes. In fig. 3, black arrows indicate focal-death cells, and spherical objects framed by red boxes indicate focal-death corpuscles.
Detailed Description
Experimental procedures according to the invention, in which no particular conditions are specified in the following examples, are generally carried out under conventional conditions, or under conditions recommended by the manufacturer. The various chemicals used in the examples are commercially available.
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 in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to only those steps or modules listed, but may alternatively include other steps not listed or inherent to such process, method, article, or device.
The "plurality" referred to in the present invention means two or more. "and/or" describes the association relationship of the associated objects, meaning that there may be three relationships, e.g., a and/or B, which may mean: a exists alone, A and B exist simultaneously, and B exists alone. The character "/" generally indicates that the former and latter associated objects are in an "or" relationship.
The embodiment provides a preparation method of umbilical cord mesenchymal stem cells, which comprises the following steps:
(1) perforating the sheet umbilical cord tissue;
(2) placing the cell apoptosis product in the hole formed in the step (1) for pre-culture;
(3) removing the cell tar-death product in the hole, and continuously culturing the obtained umbilical cord tissue;
the cell tar death product comprises at least one of cell tar death secretion, cell tar death extract and tar death cells generated after cell tar death.
The cell apoptosis is programmed cell inflammatory necrosis and is characterized by relying on the induction of caspase-1 (caspase-1) and accompanying the release of a plurality of proinflammatory factors. When the cells are burnt and die, the cell nucleuses are condensed, the chromatin is broken, and a plurality of burnt and die corpuscles are formed. Meanwhile, a small hole with the diameter of 1-2 nm is formed on the cell membrane, the integrity of the cell membrane is damaged, and various inflammatory factors including interleukin-1 beta (IL-1 beta) and interleukin-18 (IL-18) are released. Compared with the stimulation of single inflammatory factors (such as TNF-alpha, interleukin or INF-gamma) on the mesenchymal stem cells, the cell apoptosis product is used as the stimulation condition of the mesenchymal stem cells, so that more comprehensive inflammatory factor types are provided, the inflammatory environment formed after the tissues in vivo are damaged is more approximate, and the activation effect on the mesenchymal stem cells is better.
In some embodiments, the cell apoptosis product of step (2) is mixed with a gel matrix and the resulting mixture is placed into the pores. The gel matrix can release the pyrophoric inflammatory factor contained in the gel matrix into a culture medium through diffusion and has good biocompatibility. Preferably, the gel matrix is any one or combination of more than one of natural polypeptide hydrogel and natural biological gel. Preferably, the native polypeptide hydrogel is a nano-polypeptide hydrogel, and the native biogel is preferably at least one of fibrin gel, extracellular matrix gel and collagen gel.
In some embodiments, the concentration of the protein in the mixture of the cell apoptosis product obtained in step (2) and the gel matrix is 1ng/ml to 10mg/ml, preferably 20ng/ml to 1mg/ml, and the concentration is kept in a low range, so that umbilical cord mesenchymal stem cells in the tissue can be effectively activated, a driving force is provided for migration of the stem cells, and a too high concentration can inhibit the growth and proliferation of cells and even cause cell death of the cells due to a reaction which can cause excessive inflammation.
In some embodiments, the pre-culturing time in step (2) is 1-148 h, preferably 6-48 h.
In some embodiments, step (3) removes the cell apoptosis product from the wells, and replaces the wells with a new complete culture medium for further culture, and subculture is performed until 80% -90% of the cells migrated have fused.
In some embodiments, the cells of step (2) are at least one of monocytes, fibroblasts, endothelial cells, smooth muscle cells, nerve cells, cardiac muscle cells, corneal stromal cells, totipotent stem cells, pluripotent stem cells, and unipotent stem cells. For example, it may be a bone marrow mesenchymal stem cell, a limbal stem cell or a cardiomyocyte-derived cell.
In some embodiments, the method of inducing said cell to produce said cell apoptosis product comprises: physical induction, chemical induction or biological induction. The physical induction method comprises any one or more methods of ultraviolet irradiation induction, ultrasonic induction, radiation stimulation induction and hydroxypropyl cellulose caprylate coated dish culture; the chemical induction method comprises any one or more method combination of cell perforin induction, urate crystallization induction and flagellin induction; in addition, the biological induction method comprises inducing infection of the cells with any one or more microorganisms selected from the group consisting of Pseudomonas, Listeria, Shigella, Legionella, Pseudomonas aeruginosa, Francisella, Yersinia, Streptococcus pneumoniae, Actinobacillus pleuropneumoniae, Candida albicans, Staphylococcus aureus, Salmonella typhi, hepatitis virus, and immunodeficiency virus.
In some of these embodiments, the aperture is a through aperture. Preferably, the perforated area accounts for 10-90% of the whole area of the sheet umbilical cord tissue. Wherein. The diameter of the hole is 0.1cm to 10cm, the preferable range is 0.5cm to 3cm, and further preferably 0.5cm to 1.5cm, and specifically, the hole diameter may be 0.4cm, 0.5cm, 0.6cm, 0.7cm, 0.8cm, 0.9cm, 1cm, 1.1cm, or 1.2 cm. The shape of the hole is not limited, and is preferably circular or elliptical. Further preferably, the distribution density of the pores on the umbilical cord tissue is 25cm per210-60 holes are punched. Wherein, the shape and the size of each hole are independent, and each hole is separated from each other.
In some of these embodiments, the preparation of the sheet of umbilical cord tissue comprises subjecting the umbilical cord to a washing and separation step; the washing comprises performing conventional washing of the umbilical cord tissue using physiological saline or phosphate buffer with or without antibiotics. Wherein the antibiotic is one or more of chloramphenicol, streptomycin and gentamicin. The separation step is to cut the skin of the umbilical cord along the long axis of the tissue and then spread the umbilical cord to obtain the sheet umbilical cord tissue containing or not containing the artery or vein.
In some embodiments, the pre-culture comprises a fully submerged culture and a partially submerged culture. The completely immersed culture refers to that the sheet umbilical cord tissues are completely immersed below the liquid level of a culture medium; the partial immersion culture is to locate 1-99% of the sheet umbilical cord tissue below the liquid level of the culture medium, and the rest part is above the liquid level of the culture medium.
In some embodiments, the medium of said pre-culturing of step (2) and said culturing of (3) is complete medium; wherein the complete culture medium is prepared by diluting the following components: low-sugar DMEM, (10 +/-2)% (v/v) fetal bovine serum, (1 +/-0.2)% (v/v) glutamine, and (1 +/-0.2)% (v/v) penicillin-streptomycin solution.
In some of these embodiments, the source of the umbilical cord tissue is a human or non-human mammal; further, the non-human mammal includes, but is not limited to, a dog, a pig, or a horse.
The present invention will be described in further detail with reference to specific examples.
Example 1 obtaining umbilical cord mesenchymal stem cells using pyrophorome cultured from mesenchymal stem cells
(1) Obtaining sheet-like umbilical cord tissue
Shaking and cleaning the obtained umbilical cord tissue for 3-4 times by using Phosphate Buffered Saline (PBS) containing 1% (v/v) penicillin and streptomycin, cutting off the outer skin of the umbilical cord tissue along a vein by using a tissue cutter, and removing two arteries and a vein by using tissue forceps to obtain a piece of umbilical cord tissue with the side length of 5 cm.
(2) Punching
In a clean bench, a circular punch with a diameter of 0.5cm was used, and the area was 25cm2The sheet-shaped umbilical cord tissue is evenly perforated with 40 through holes.
(3) Acquisition of inflammatory environment of cell scorching
Bone marrow mesenchymal stem cells were cultured at 1 × 106 cells/cm2The cells were cultured for 3 days in OPC (hydroxypropyl cellulose caprylate) coated dishes to form distinct apoptotic bodies (as shown in FIG. 3), and then the medium containing inflammatory factors of apoptosis was collected. The collected culture medium is filtered through a filter membrane of 0.22 mu m to remove cell debris, and the cell apoptosis secretion is obtained. The protein concentration of the secretion of the apoptotic cells was measured using BCA kit, and the concentration was adjusted to 0.4mg/ml with sterile PBS. Matrigel (belonging to extracellular matrix gel, purchased from
CATALOG NUMBER:356234) was mixed with the prepared cell apoptosis secretion at a volume ratio of 1:1, and left at room temperature to wait for its coagulation.
(4) Local pyrophoric death inflammatory environment pre-culture
Cutting the matrigel coagulated and mixed with the cell apoptosis secretion by using a trephine with the diameter of 0.5cm to obtain the cylindrical matrigel mixed with the cell apoptosis secretion. The perforated umbilical cord tissue is placed in a culture dish, the matrix glue mixed with the cell apoptosis secretion is filled into the pore diameter of the umbilical cord tissue correspondingly (as shown in figure 1), and meanwhile, a complete culture medium which is low-sugar DMEM + 10% (v/v) fetal bovine serum + 1% (v/v) glutamine + 1% (v/v) penicillin-streptomycin solution (100X) is added into the culture system, so that 50% of the thickness of the umbilical cord tissue is placed below the liquid level. Placing in an environment with the temperature of 37 ℃ and the carbon dioxide content of 5 percent for pre-culture, wherein the pre-culture time is 12 h.
(5) Culturing and passaging in normal environment.
After pre-culture, the matrigel containing the inflammatory factor of apoptosis was removed from the pore size and replaced with fresh complete medium. And carrying out passage until 80-90% of the migrated cells are fused. Cells migrate after 12-24 hours after the cells are pre-cultured, the cell fusion rate reaches 90-95% 1-2 days after the pre-culture, the survival rate of the obtained umbilical cord mesenchymal stem cells is 95% through detection, and subculture can be carried out (as shown in figure 2). Compared with the method that the cells are migrated after the tissue adherent primary culture for 7 days, the method greatly improves the migration rate of the umbilical cord mesenchymal stem cells.
Immunophenotyping analysis: the detection by flow cytometry shows that the cells highly express CD29, CD105, CD44 and CD73, and have extremely low expression of CD31, CD34, CD45 and MHC-II.
Differentiation potential identification:
adipogenic differentiation induction results: after 14 days of directional induction, the umbilical cord mesenchymal stem cells obtained in the embodiment can be seen to be strongly positive by oil red O staining, and the cytoplasm can be seen to contain red lipid droplets under an inverted microscope.
Osteogenic differentiation induction results: after 14 days of directional induction, in the test process of the umbilical cord mesenchymal stem cells obtained in the embodiment, the cells are full of black particles and are not uniform in size.
The above results indicate that the umbilical cord mesenchymal stem cells isolated in this example have good osteogenic differentiation capacity for lipid formation.
Detecting the paracrine capacity of cells: the ELISA and qPCR are used for detecting the gene expression and paracrine action of VEGF, bFGF and HGF, and the detection result shows that the expression of the VEGF, bFGF and HGF genes of the umbilical cord mesenchymal stem cells separated in the embodiment is obviously increased, and the concentration of VEGF, bFGF and HGF cell growth factors in a culture solution is obviously increased compared with that of primary mesenchymal stem cells obtained by normal adherence. The biological activity of the umbilical cord mesenchymal stem cells prepared by the embodiment is obviously improved.
Example 2 obtaining umbilical cord mesenchymal stem cells by using pyrophoric corpuscles cultured by limbal stem cell pyrophoric death
(1) Obtaining sheet umbilical cord tissue
Shaking and cleaning the obtained umbilical cord tissue for 3-4 times by using Phosphate Buffered Saline (PBS) containing 1% (v/v) penicillin and streptomycin, cutting off the outer skin of the umbilical cord tissue along a vein by using a tissue cutter, removing two arteries and a vein by using tissue forceps, cleaning again to remove blood clots adhered to the surface of the tissue, and obtaining the sheet umbilical cord tissue with the side length of 6 cm.
(2) Punching
In a clean bench, makeUsing circular punches with diameters of 0.5cm and 1cm, alternating with each other, in an area of 16cm2The sheet umbilical cord tissue of (1) is uniformly perforated with 10 through holes, wherein 5 holes are 0.5mm in diameter and 5 holes are 1cm in diameter.
(3) Acquisition of inflammatory environment of cell scorching
Limbal stem cells were plated at 1 × 106 cells/ml2The density of (b) was inoculated in a large dish having a diameter of 10cm, and cultured in a carbon dioxide incubator at 37 ℃ for 24 hours in a medium of high-glucose DMEM + 10% (v/v) fetal bovine serum + 1% (v/v) glutamine + 1% (v/v) NEAA + 1% penicillin-streptomycin (100X) + 0.02% (v/v) EGF + 0.1% (v/v) transferrin + 0.1% (v/v) insulin. After 24h, the medium was changed and the medium was changed at 80mJ/cm2Ultraviolet irradiation for 4 hours. Culturing for 24h after irradiation, collecting culture solution after culturing, washing with sterile PBS for three times, gently blowing, blowing off cell surface substances, collecting PBS, ultracentrifuging collected culture medium and PBS at 12000rpm, removing supernatant, and collecting apoptotic bodies. The concentration of apophotosomal protein was measured using BCA kit, adjusted to 100ng/ml with sterile PBS, and mixed with collagen gel at a volume ratio of 1: 1.
(4) Local coke-death environment pre-culture
The collagen gel containing the pyrogen-free bodies was drilled into 5 cylinders each having the pyrogen-free bodies using trephines having a diameter of 0.5mm and 1 cm. The cylindrical collagen gel containing the pyrophoric bodies is filled into the pore size of the umbilical cord tissue correspondingly, and meanwhile, a complete culture medium which is low-sugar DMEM + 10% (v/v) fetal bovine serum + 1% (v/v) glutamine + 1% (v/v) penicillin-streptomycin solution (100X) is added into the culture system, so that 20% of the thickness of the umbilical cord tissue is placed below the liquid level. Placing the mixture in an environment with the temperature of 37 ℃ and the carbon dioxide content of 5 percent for pre-culture, wherein the pre-culture time is 24 hours.
(5) Culturing and passaging in normal environment.
After preculture, collagen gel containing pyrophoric bodies was removed from the pore size and replaced with fresh complete medium. And carrying out passage until 80-90% of the migrated cells are fused. After the cells are pre-cultured and cultured for 36h, the cells migrate out, the migration rate of the cells in 10 holes is 100% (migration rate is the number of the holes with the cells migrated out/the total pore diameter number), the fusion rate of the cells reaches 90% after the cells are pre-cultured for 48h, and subculture can be carried out.
Cell migration ability test:
the primary cells obtained by the method of the invention and the primary cells obtained by the conventional tissue digestion method are all 3 × 105 cells/cm2The density of the culture medium is inoculated in a six-hole plate, a straight line is vertically and smoothly drawn out by using a gun head of a 1ml liquid transferring gun to be clung to the bottom of the six-hole plate after 24 hours, and the culture medium is replaced after drawing. And photographing and recording under a mirror at 0h, 6h and 12h after scratching, and evaluating the migration rate by using Image pro-Plus software to show that the primary cells obtained by the method have the fastest migration rate, the closing of the scratches is realized within 12h, and the primary cells obtained by a tissue digestion method can realize the closing of the scratches within 48 h.
And (3) detecting the inflammation inhibition capacity:
the primary umbilical cord mesenchymal stem cells obtained by the method and the primary umbilical cord mesenchymal stem cells obtained by the conventional tissue digestion method are respectively pre-stimulated with the lipopolysaccharide according to the ratio of 10: 1, collecting culture medium supernatant after culturing for 18h, and detecting the concentration of TNF-alpha in the culture medium by using an ELISA kit of the TNF-alpha, wherein the result shows that compared with the primary cells obtained by a conventional tissue complete digestion method, the primary cells obtained by the method can obviously reduce the concentration of the TNF-alpha in the culture medium, and the umbilical cord mesenchymal stem cells obtained by the method obviously improve the capacity of inhibiting lymphocytes to secrete the TNF-alpha.
Example 3: preparation of mesenchymal stem cells by using myocardial sphere-derived cell apoptosis secretion
(1) Obtaining sheet umbilical cord tissue
Shaking and cleaning the obtained umbilical cord tissue for 3-4 times by using Phosphate Buffered Saline (PBS) containing 1% (v/v) penicillin and streptomycin, cutting off the skin of the umbilical cord tissue along veins by using a tissue cutter, cleaning again to remove blood clots adhered to the surface of the tissue, and obtaining the umbilical cord tissue with an area of 24cm2The sheet-like umbilical cord tissue of (1).
(2) Punching
In a clean bench, a 12mm diameter circular punch was used, at an area of 24cm2The sheet umbilical cord tissue of (1) is uniformly perforated with 12 through holes.
(3) Acquisition of inflammatory environment of cell scorching
Obtaining cardiomyocyte-derived cells, and adding the cells to the culture medium at 2 × 104cells/cm2Inoculating to OPC (hydroxypropyl cellulose caprylate) coated dish, culturing for 3 days, observing the state in the dish, sucking culture supernatant until corpuscles appear, part of cell forms become egg-fried samples and cells become scorched, filtering with a 0.22 μm filter, adjusting the concentration to 50ng/ml with sterile PBS, and freezing and storing in a 4 ℃ refrigerator for a short time for later use.
(4) Local coke-death environment pre-culture
Mixing the filtered culture medium and the nano-polypeptide hydrogel in a volume ratio of 1:1, and filling the mixture into the pore size of the umbilical cord tissue. Adding a complete culture medium into the culture system, wherein the complete culture medium is low-sugar DMEM + 10% (v/v) fetal bovine serum + 1% (v/v) glutamine + 1% (v/v) penicillin-streptomycin solution (100X), placing one third of the thickness of the umbilical cord tissue below the liquid level of the culture solution, placing the umbilical cord tissue in a thermostatic incubator with 37 ℃ and 5% carbon dioxide for inflammatory stimulation preculture (A group), and the culture time is 12 h.
(5) Culturing and passaging in normal environment.
After pre-stimulation culture, the mixed nano polypeptide hydrogel is removed from the pore size, and a fresh complete culture medium is replaced. And carrying out passage when the cells of the umbilical cord tissue are migrated out to reach the cell fusion rate of 80-90% of the culture substrate. Cell migration time was observed and compared to tissue blocks not pre-incubated with apoptosis factor (group B). The A group umbilical cord mesenchymal stem cells migrate after 12 hours of pre-stimulation culture, the cell fusion rate reaches 95% after 2 days of pre-stimulation culture, and subculture can be carried out. After 6 days, the B group umbilical cord mesenchymal stem cells are emigrated, and after 10 days, the cell fusion rate reaches 90%. Both groups of cells grew in long spindle, vortex shapes.
And (3) detecting stress resistance:
the primary cells obtained by the method of the invention are divided into 1 × 105 cells/ml2Inoculating in a large dish, and addingAdding a culture medium containing TNF-alpha with the concentration of 500ng/ml, culturing for 24h, and identifying the cell survival rate through dead-live staining, wherein the survival rate of the group A is 90%, and the survival rate of the group B is 70%.
And (3) detecting paracrine capacity:
a, B two groups of cells were tested for paracrine effect by qPCR, and the transcript levels of HGF, VEGF, IGF, and bFGF in group A were all higher than those in group B.
In conclusion, the proliferation capacity, the anti-adversity capacity and the paracrine capacity of the umbilical cord mesenchymal stem cells obtained by the method are obviously improved.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A preparation method of umbilical cord mesenchymal stem cells is characterized by comprising the following steps:
(1) perforating the sheet umbilical cord tissue;
(2) placing the cell apoptosis product in the hole formed in the step (1) for pre-culture;
(3) removing the cell tar-death product in the hole, and continuously culturing the obtained umbilical cord tissue;
the cell tar death product comprises at least one of cell tar death secretion, cell tar death extract and tar death cells generated after cell tar death.
2. The preparation method according to claim 1, wherein the perforated area accounts for 10-90% of the whole area of the sheet-shaped umbilical cord tissue; and/or the diameter of the hole is 0.1 cm-10 cm, preferably 0.5-3 cm; and/or, the hole is a through hole.
3. The method of claim 1, wherein the cell apoptosis product of step (2) is mixed with a gel matrix and the resulting mixture is placed in the well.
4. The method of claim 3, wherein the gel matrix is at least one of a native polypeptide hydrogel and a native biogel; preferably, the natural polypeptide hydrogel is a nano polypeptide hydrogel, and the natural biological gel is at least one of fibrin gel, extracellular matrix gel and collagen gel.
5. The method according to claim 3, wherein the protein concentration of the mixture of the cell apoptosis product of the step (2) and the gel matrix is 1ng/ml to 10 mg/ml; and/or the pre-culture time in the step (2) is 1-148 h, preferably 6-48 h.
6. The method according to claim 1, wherein the cells of step (2) comprise at least one of monocytes, fibroblasts, endothelial cells, smooth muscle cells, nerve cells, cardiac muscle cells, corneal stromal cells, totipotent stem cells, pluripotent stem cells and unipotent stem cells.
7. The method of any one of claims 1 to 6, wherein the method of inducing the production of the apoptosis product comprises: physical induction, chemical induction or biological induction.
8. The preparation method according to claim 7, wherein the physical induction method comprises any one or more of ultraviolet irradiation induction, ultrasonic induction, radiation stimulation induction, hydroxypropyl cellulose caprylate coated dish culture; and/or, the chemical induction method comprises any one or more method combination of cell perforin induction, urate crystallization induction and flagellin induction; and/or, the biological induction method comprises inducing infection of the cells by using any one or more than one microorganism selected from pseudomonas, listeria, shigella, legionella, pseudomonas aeruginosa, francisella, yersinia, streptococcus pneumoniae, actinobacillus pleuropneumoniae, candida albicans, staphylococcus aureus, salmonella typhi, hepatitis virus and immunodeficiency virus.
9. The method according to claim 1, wherein the pre-culture means includes a full submerged culture and a partial submerged culture; and/or, the pre-culture of step (2) and the culture medium of step (3) is a complete medium; and/or, the source of the umbilical cord tissue is a human or non-human mammal; further, the non-human mammal includes a dog, pig, or horse; and/or, the sheet-like umbilical cord tissue comprises a post-skinned, deployed umbilical cord tissue with or without arteries or veins.
10. The mesenchymal stem cell prepared by the preparation method of any one of claims 1 to 9.
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