CN115216441B - Composite scaffold for three-dimensional culture of stem cells and preparation method thereof - Google Patents
Composite scaffold for three-dimensional culture of stem cells and preparation method thereof Download PDFInfo
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- CN115216441B CN115216441B CN202210926837.3A CN202210926837A CN115216441B CN 115216441 B CN115216441 B CN 115216441B CN 202210926837 A CN202210926837 A CN 202210926837A CN 115216441 B CN115216441 B CN 115216441B
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2513/00—3D culture
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
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- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
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- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Developmental Biology & Embryology (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
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- Microbiology (AREA)
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- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a composite scaffold for three-dimensional culture of stem cells, which is formed by taking apple fruits subjected to decellularization treatment as a framework and crosslinking the apple fruits with a pig liver decellularized matrix. The composite scaffold has less residual cell nucleuses and tissue DNA, high content of collagen fibers and cellulose, high porosity and water absorption rate, no cytotoxicity, is an ideal scaffold material, can promote adhesion and proliferation of stem cells, and remarkably increases the expression level of the cultured cat adipose mesenchymal stem cells TSG-6, TGF-beta and EGF cytokines.
Description
Technical Field
The invention relates to a composite scaffold for three-dimensional culture of stem cells, and also relates to a preparation method and application of the composite scaffold, belonging to the field of biology.
Background
Cells are the basic material of all research experiments, and different culture modes have different influences on experimental results. Cells grown in two dimensions grow in a flat dispersion, lacking support in the vertical direction, resulting in tip-substrate polarity. The production of polarity, although normal for certain Cell types, two-dimensional cultured cells are forced into unnatural states, which are greatly different from those grown in the real environment in vivo, may exhibit altered functions and phenotypes, limiting the development of many studies, such as fields of drug screening, cell growth, apoptosis, and expression of gene proteins (McBeath, R, pirone, D M and Nelson, C M, et al Cell shape, cytoskeletal tension, and RhoA regulate stem Cell lineage com-munity, dev Cell,2004,6 (4): 483-495). The three-dimensional culture technology greatly simulates the internal environment of the organism, and has greatly advanced in the field of tissue regeneration medicine to date. Three-dimensional culture techniques can be divided into two kinds of techniques, namely scaffold-dependent and scaffold-free techniques, the former mainly comprises natural tissue decellularized matrix, hydrogel prepared from polysaccharides, porous scaffolds similar to scaffolds prepared from different processes, microcarriers and microfluidic culture techniques. The latter, independent of biological materials, spontaneously aggregates into spheres from cells, forming a three-dimensional structure. Among them, the special matrix structure is more favorable for cell adhesion proliferation and maintenance of cell differentiation state due to excellent biocompatibility and low immunogenicity of natural decellularized cell matrix, and thus is used in various studies.
Liver disease is a major cause of disease and death worldwide (Wang, F S, fan, J G and Zhang, Z, et al, global burden of liver disease: the major impact of China, hepatology,2014,60 (6): 2099-2108.) the gold standard for current treatment of end-stage liver disease is liver transplantation, but liver source is scarce, and there are associated risks of post-transplantation rejection and long-term sequelae associated with immunosuppression (Astani, S K, devarbhavi, hand Eaton, J, et al, burden of liver diseases in the world J hepatil, 2019,70 (1): 151-171.; dienstag, J L and Cosimi, AB. Liver trans-displacement- -a vision reduction, engl J Med,2012,367 (16): 1483-1485.). With the rapid development of tissue engineering, researchers began to direct their eyes to the application of the liver acellular matrix. After the whole organ decellularization of the heart in 2008 Ott et al (Ott, H C, matthiesen, T S and Goh, S K, et al, fusion-decellularized matrix: using natural' S platform to engineer a bioartificial heart. Nat Med,2008,14 (2): 213-221), a hot blast for the whole organ decellularization was raised.
Recent progress in the decellularized matrix research of liver has been rapid, for example, uygun et al first uses SDS to decellularize its entire liver through ischemic rat portal vein and reseedes perfusion cultured mature hepatocytes to provide proof of principle for the generation of transplantable liver grafts as potential treatment methods for liver disease (Uygun, B E, soto-Gutierrez, aand Yagi, H, et al, organic reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix. Nat Med,2010,16 (7): 814-820.); pan et al have found that preparing an in vivo decellularized matrix of a single lobe and reconstructing the lobe by decellularizing provides a practical and unique technique (Pan, J, yan, S and Gao, J J, et al, in-vivo organ engineering: perfusion of hepatocytes in a single liver lobe scaffold of living rates.int J Biochem Cell Biol,2016, 80:124-131.); the pig liver and the human liver are more similar in shape, are cheap and easy to obtain, and are the optimal scaffold material for constructing the in vitro tissue engineering liver at present in terms of clinical treatment significance. However, the whole organ structure is complex, and the whole organ decellularization method of rodents is time-consuming and material-consuming, and the microstructure in the viscera is difficult to be destroyed. To solve this problem, researchers have expanded the use of decellularized scaffolds. The preparation of its ECM in powder form after lyophilization has been studied, the granular form of ECM being of interest for injection into tissues and the development of three-dimensional scaffolds (Park, K M, hussein, K H and Hong, S H, et al decellularised Liver Extracellular Matrix as Promising Tools for Transplantable Bioengineered Liver Promotes Hepatic Lineage Commitments of Induced Pluripotent Stem cells.tissue en g Part a,2016,22 (5-6): 449-460.); lonker et al used liver from different species and used the prepared decellularized matrix digest as a supplement to cultured hepatocytes, and experimental results showed that the lysed pig liver decellularized matrix had the potential to enhance drug discovery culture systems, liver whole organ engineering and hepatocyte transplantation therapies (Loneker, AE, faulk, D M and Hussey, G S, et al, solubilized liver extracellular matrix maintains primary rat hepatocyte phenotype in-vitro.J Biomed Mater Res A,2016,104 (4): 957-965.).
Cellulose sources are abundant and available from bacteria, fungi, algae, tunicates and plants, are easy to produce and meet any standard for biological materials. Compared with a scaffold synthesized by manpower, the natural cellulose scaffold is difficult to achieve on the fine standard regulation, but better in biocompatibility, simpler in preparation process, short in time consumption and low in cost. For example, modulevsky et al used cheaper apples for decellularization treatment to prepare natural cellulose scaffolds and inoculated with three mammalian cells, and experimental results showed that the cells could be adhered and proliferated in the scaffolds, and that the cells remained high in viability even after 12 consecutive culture periods, even reaching cell densities comparable to other natural and synthetic scaffolds, and that the apple decellularized cellulose scaffolds were modified with glutaraldehyde and porcine type I collagen, respectively, to control porosity and increase seeding efficiency, (Modulevsky, D J, lefebvre, C and Haase, K, et al, apple derived cellulose scaffolds for 3D mammalian cell culture.PLoS One,2014,9 (5): e 97835.). However, the time required from the inoculation of the animal cells on the apple-derived cellulose scaffold to the complete invasion is too long, the substances used for modifying the apple-derived cellulose scaffold in the existing literature are relatively single, and the related research reports on three-dimensional cultured animal stem cells are rarely seen, so that the whole organ decellularized matrix modified apple-derived cellulose scaffold can become a novel potential scaffold for culturing animal stem cells in the future.
Disclosure of Invention
The first object of the invention is to provide a composite scaffold for three-dimensional culture of stem cells, which is formed by taking apple fruits subjected to decellularization treatment as a framework and crosslinking the apple fruits with a pig liver decellularized matrix, wherein the surface of the composite scaffold is rugged, and the pig liver decellularized matrix is covered on the surface layer of the framework and permeates into the loose and porous interior of the framework.
A second object of the present invention is to provide a method for preparing the composite scaffold, comprising the steps of:
(1) Sequentially carrying out cell removal treatment on the pork liver by using EDTA, SDS and DNase I enzymes, freeze-drying and crushing the treated pork liver to obtain a pork liver cell removal matrix;
(2) Cutting apples into pieces, performing cell removal treatment by SDS (sodium dodecyl sulfate), and freeze-drying to obtain a framework;
(3) Adding the pig liver acellular matrix and pepsin into a dilute hydrochloric acid solution, stirring to form a uniform solution, regulating the pH of the solution to 7-8, dialyzing to prepare a pig liver acellular matrix solution with the concentration of 2-9mg/mL, adding the acellular treated apple fruits, simultaneously adding EDC and NHS, soaking at room temperature, performing ultrasonic treatment, and cleaning to obtain the pig liver acellular matrix.
Preferably, in step (3), the pH of the solution is 7.4.
Preferably, in the step (3), the concentration of the pig liver acellular matrix solution is 5mg/mL.
Preferably, in step (3), the molar ratio of EDC to NHS is 1:1.
preferably, in the step (3), the ultrasonic frequency is 50-200KHZ, and the time is 10-60min.
Preferably, the concentration of EDTA, SDS, DNase I type enzyme is 0.02%, 5%, 0.01%, respectively.
According to one embodiment of the invention, one of the best methods of preparation is:
(1) Sequentially carrying out decellularization treatment on the pork liver by using 0.02% EDTA, 5% SDS and 0.01% DNase I type enzyme, freeze-drying and crushing the treated pork liver to obtain a pork liver decellularized matrix;
(2) Cutting apples into pieces, performing cell removal treatment with 5% SDS at a treatment temperature of 4 ℃ for 24 hours, and freeze-drying the apples to be used as a framework after the treatment;
(3) Adding 250 parts by weight of pig liver acellular matrix and 80 parts by weight of pepsin into a dilute acid solution, stirring to form a uniform solution, regulating the pH of the solution to 7.4, dialyzing to prepare a pig liver acellular matrix solution with the concentration of 5mg/mL, adding acellular treated apple fruits, and simultaneously adding a molar ratio of 1:1, soaking the EDC and the NHS for 4 hours at room temperature, then carrying out ultrasonic treatment with the ultrasonic frequency of 120KHZ for 30 minutes, and cleaning after ultrasonic treatment.
The composite scaffold provided by the invention has the advantages of less residual cell nucleuses and tissue DNA, high content of collagen fibers and cellulose, high porosity and water absorption rate, no cytotoxicity and is an ideal scaffold material. The composite scaffold can promote the adhesion and proliferation of stem cells, and the expression quantity of the cat adipose mesenchymal stem cells TSG-6, TGF-beta and EGF cytokines cultured by the composite scaffold is obviously increased.
Drawings
Fig. 1: a composite scaffold structure under a scanning electron microscope, (a) an apple scaffold of uncrosslinked pig liver decellularized matrix; (B) a composite scaffold for cross-linking the acellular matrix of the pig liver.
Fig. 2: histomorphology staining observation of pig liver acellular matrix, (A) control group pig liver HE staining; (B) control pig liver Masson staining; (C) pig liver DAPI staining of control group; (D) treating group pig liver HE staining; (E) treatment group pig liver Masson staining; (F) DAPI staining of pig livers in the treatment group.
Fig. 3: detecting the DNA content of the pig liver acellular matrix and agarose gel electrophoresis, (A) detecting the residual DNA content; (B) agarose gel electrophoresis.
Fig. 4: quality detection of apple decellularized scaffold, (a) safranin-fast green staining of untreated apple tissue; (B) safranin-fast green staining of treated apple tissue; (C) comparing the DNA content of apple tissues before and after decellularization; (D) quality comparison of apple decellularized scaffold before and after water absorption. Scale bar: 100 μm. Data are expressed in mean±sd, which represents statistical significance compared to Control group, P < 0.0001.
Fig. 5: influence of the composite scaffold on proliferation of feline adipose mesenchymal stem cells.
Fig. 6: the survival rate (green is living cells, red is dead cells) of the cat adipose mesenchymal stem cells cultured and fixed and dyed in the composite scaffold is observed under a fluorescence microscope, and the ratio scale is: 50 μm.
Fig. 7: and observing the cat adipose mesenchymal stem cell skeleton cultured and fixed and dyed in the composite bracket under a laser confocal microscope. (A) Proliferation depth of cat adipose mesenchymal stem cells in the scaffold; (B) Maximum projection of adhesion state of cat adipose-derived mesenchymal stem cells in a bracket, and a scale: 50 μm.
Fig. 8: influence of composite scaffolds on expression of three cytokines after culture of cat adipose-derived mesenchymal stem cells, in the figure, group ABCD represents apple decellularized cytoskeleton without composite pig liver decellularized matrix, composite scaffold prepared in example 3, composite scaffold prepared in example 2, and composite scaffold prepared in example 1, respectively. * P <0.05, P <0.01.
Detailed Description
The present invention will be described in detail with reference to the following examples.
Example 1
1. Method for decellularizing pig liver by combining biological enzyme and chemical reagent
(1) Preparing tissue decellularization liquid I (0.02% EDTA): 0.6g of ethylenediamine tetraacetic acid disodium salt (EDTA), 800mL of 0.9% sodium chloride, and then fixing the volume to 3000mL, and filtering with a 10mL syringe and a filter for later use;
(2) Preparing tissue decellularization liquid II (5% SDS): 75g of Sodium Dodecyl Sulfate (SDS), firstly using 800mL of 0.9% sodium chloride to fully dissolve until bubbles are completely removed, then adjusting the PH value to 7.5, and then fixing the volume to 1500mL for filtration for later use;
(3) Preparing tissue cell removing liquid III (0.01% DNase I type enzyme): 0.1g DNase I enzyme, using 800mL,0.9% sodium chloride fully mixing, then constant volume to 1000mL, filtering and split charging for standby;
(4) Washing fresh pork liver which is just purchased with clear water until no blood water is seen, washing with double distilled water for 10 times, blocking, and freezing in an ultralow temperature refrigerator at-80 ℃ for about 1-2 hours;
(5) Fixing and cutting the semi-unfrozen pig liver into liver slices with the thickness of 2mm by a slicer, putting the liver slices into a special barrel of an electric stirrer at the rotating speed of 520r/min, adding tissue decellularization liquid I with the volume twice that of the pig liver, stirring at room temperature for 12 hours, and then washing with double distilled water until the liquid has no blood color;
(6) Discarding double distilled water, adding tissue decellularized liquid II with the volume twice that of the pork liver, stirring for 12h (the tissue decellularized liquid II can be properly prolonged or shortened according to the transparency condition of liver slices), and then washing with the double distilled water until the pork liver is completely submerged to the bottom of the barrel;
(7) Discarding the cleaning solution in the previous step, adding the tissue cell removal solution III with the same volume, stirring for 12 hours, and then stirring, cleaning and changing water for at least 10 times by using 1X PBS after autoclaving;
(8) Spreading the processed pig liver acellular matrix into a sterilizing plate, sealing a film, punching holes with needles, and freeze-drying in a freeze dryer for 48h;
(9) Grinding the lyophilized matrix of pig liver decellularized with tissue superfine pulverizer into powder, and storing at-80deg.C.
The pork liver cut after thawing and before cell removal is bright red; pig liver slices treated with EDTA solution appear non-uniformly dark red; after SDS and DNase I enzyme treatment, the liver lobule structure is obvious, the color is completely whitened, and the liver lobule has certain elasticity; the freeze-dried pig liver acellular matrix presents flocculent; the ground pig liver acellular matrix is in uniform particles.
2. Preparation of apple decellularized cellulose skeleton
(1) Putting the purchased fresh ripe rock candy apples into a refrigerator at the temperature of minus 20 ℃ for freezing for 5min;
(2) Cutting the apple into blocks with the thickness of 2mm by using a slicer, taking care that the apple middle ovary wall is not cut;
(3) Putting the apple slices into 5% SDS (in step 1), and performing decellularization treatment for 24 hours by magnetic stirring at 4 ℃ and 220 r/min;
(4) Discarding SDS, and repeatedly cleaning the acellular apple bracket by using the sterilized PBS;
(5) And fishing out the cleaned bracket and freeze-drying.
The cut apple fruit tissue presents light yellow; after SDS decellularization treatment, the jelly is semitransparent; a large number of pinholes were visible to the naked eye after lyophilization.
3. Preparation of composite scaffold
(1) Preparation of pig liver acellular matrix solution
Taking freeze-dried pig liver acellular matrix powder, sieving with a 80-mesh steel sieve, taking 250mg of the sieved powder, adding 50mg of pepsin into 0.01M dilute hydrochloric acid solution, stirring and digesting for 72h to form uniform solution; then adjusting the pH of the solution to about 7.4 to terminate the digestion of pepsin; preparing a 1000Da dialysis bag, cutting into proper length, boiling for 15min, washing with double distilled water for 3 times, pouring the solution into the dialysis bag, putting the double distilled water into the dialysis bag for dialysis for 48 hours, and taking out to prepare the pig liver acellular matrix solution with the concentration of 5mg/mL.
The prepared pig liver acellular matrix solution presents semitransparent colloid.
(2) Pig liver decellularized matrix coated apple-derived cellulose stent
Taking freeze-dried apple decellularized cellulose skeleton, adding into a prepared pig liver decellularized matrix solution with the concentration of 5mg/mL, soaking, and simultaneously adding EDC and NHS (the molar ratio of the EDC to the NHS is 1/1). After soaking for about 4 hours at room temperature, the uncrosslinked matrix is washed by PBS with ultrasonic crosslinking for 30 minutes and ultrasonic frequency of 120KHZ until white granular substances no longer exist in the PBS, and the preparation of the composite scaffold is completed.
The surface of the prepared composite scaffold is extremely rugged, and the pig liver acellular matrix is observed to cover the surface layer of the apple cellulose scaffold and penetrate into the loose and porous interior of the apple cellulose scaffold under a 100X scanning electron microscope (figure 1).
Example 2
In this example, the preparation method of the pig liver decellularized matrix and the apple decellularized cellulose skeleton is the same as that of example 1, and the preparation method of the composite scaffold is as follows:
grinding and sieving pig liver acellular matrix, taking 250mg of powder, adding 150mg of pepsin into 0.01M dilute hydrochloric acid solution, stirring to form uniform solution, regulating the pH of the solution to 7.4, dialyzing to prepare pig liver acellular matrix solution with the concentration of 8mg/mL, adding apple acellular cellulose skeleton, simultaneously adding EDC and NHS, soaking at room temperature for about 5 hours, performing ultrasonic treatment with the ultrasonic frequency of 150KHZ for 20 minutes, and washing with PBS to obtain the pig liver acellular matrix.
Example 3
In this example, the preparation method of the pig liver decellularized matrix and the apple decellularized cellulose skeleton is the same as that of example 1, and the preparation method of the composite scaffold is as follows:
grinding and sieving pig liver acellular matrix, taking 250mg of powder, adding 100mg of pepsin into 0.01M dilute hydrochloric acid solution, stirring to form uniform solution, regulating the pH of the solution to 7.4, dialyzing to prepare pig liver acellular matrix solution with the concentration of 3mg/mL, adding apple acellular cellulose skeleton, simultaneously adding EDC and NHS, soaking at room temperature for about 2 hours, performing ultrasonic treatment with the ultrasonic frequency of 200KHZ for 10min, and cleaning with PBS to obtain the pig liver acellular matrix.
Test examples
1. Detection of histological, biochemical and physical properties of pig liver acellular matrix
The currently internationally accepted acellular matrix generally satisfies the following three conditions: first, residual DNA concentrations were less than 50ng/mg ECM; secondly, the length of the residual DNA fragment is less than 200bp; finally, no nuclei are visible in the tissue.
HE staining, hematoxylin and eosin staining, the former stained the nucleus blue, the latter stained the intracellular eosinophil (bright red), collagen fiber (pale pink), spandex fiber (bright pink) to varying degrees of red (fig. 2-a), whereas pig liver was not seen after cell removal (fig. 2-D). In addition, masson staining also allowed detection of matrix quality, with pig liver collagen fibers being blue, cytoplasm being red, and nuclei being black-blue (FIG. 2-B), whereas most of the analyses were collagen fibers as seen by the decellularized matrix binding HE (FIG. 2-E). After DAPI staining, a large number of dark blue nuclei were detected in porcine liver tissue (FIG. 2-C), and only a small amount of residual nuclear debris was visible in the decellularized matrix (FIG. 2-F). In conclusion, the pig liver acellular matrix reaches the standard in terms of histomorphology.
It has been reported that the residual DNA in tissues causes immune rejection when transplanted into a recipient, and therefore, the content of the residual DNA is one of the criteria for measuring whether cells are completely removed, and although the subject is not to directly transplant the pig liver acellular matrix into the body, considering that cell secretions prepared into new scaffolds for later culture are injected into animals, the content of the residual DNA needs to be detected, and compared with untreated groups, the results are shown to meet the accepted criteria (FIG. 3-A); for the same purpose, the length of the DNA residual fragment was detected by agarose gel electrophoresis, and it was found by observing (FIG. 3-B) that the DNA fragment of untreated pig liver was about 10Kb or more, whereas the decellularized pig liver had no obvious DNA fragment residual, indicating that the in vivo transplantation standard was reached.
2. Quality detection of apple decellularized cellulose scaffold
Safranin-fast green staining is a method commonly used for observing structural morphology in plant tissues. The cell nuclei and lignified cell walls were bright red, the keratinocyte walls were transparent pink, and the cytoplasm and cellulose-containing cell walls were blue-green after staining the apple fruit tissue sections (FIG. 4-A); after the acellular treatment, no residual cell nucleus is confirmed by observation (figure 4-B), and the whole bracket is basically cellulose in cell walls after the apple fruits are acellular, so that the requirement of the subject on the preparation of the composite bracket is met. The apple scaffold after lyophilization has low DNA content (figure 4-C), and meets the quality standard of acellular matrix. The high porosity is one of the conditions of an ideal scaffold, wherein the higher the water absorption rate is, the higher the porosity is reflected, the difference between the water absorption rate and the mass change before and after water absorption of the scaffold is extremely remarkable (figure 4-D), the water absorption rate is calculated to be about 400%, and the comprehensive analysis of the results proves that the scaffold meets the requirements of an ideal modifiable scaffold material.
3. Toxicity detection of composite scaffolds
After the prepared scaffolds were sterilized, they were immersed in a low sugar medium containing 10% FBS and 1% diabody for overnight incubation, and then the extract was used to culture cat adipose mesenchymal stem cells (Feline-ADMSCs). We observe the proliferation state of cells in 24h, 48h and 72h successively, and find that the cells after 24h grow well on the wall and take on irregular shape or shuttle shape (figure 5-A); shuttle cell increase after 48h, occasional oval cell floating (FIG. 5-B); compared with the first two days, the clostridial cells grown more densely after 72 hours already started to have a tendency to appear spiral (FIG. 5-C), and the overall three-day continuous observation of the cell growth state can preliminarily determine that the scaffold is nontoxic. The absorbance value of each group of cells at 490nm is detected by CCK8 and is substituted into a cell proliferation rate formula to calculate that the cell proliferation rate is more than 99%, so that the toxicity of the scaffold can be finally determined to be used for the subsequent cell inoculation experiment.
4. Proliferation and adhesion of cells within a composite scaffold
Calcein-AM can stain living cells to make them appear green fluorescence; propidium Iodide (PI) stains dead cells and fluoresces red. The method is used for detecting the living and dead conditions of cells in the three-dimensional scaffold, and the cells are observed under a fluorescence microscope after being stained. However, since the stent has a certain thickness and the material of the stent is semitransparent, when in staining observation, we can only see the living and dead condition of a small part of cells on the surface of the stent, and the surface of the crosslinked stent presents uneven condition, so that the observed cells are very small. As shown in fig. 6, green shows the shape of surviving cat adipose mesenchymal stem cells, and the sparse distribution of dead cells, although both were rare, it was confirmed that the number of viable cells was far higher than that of dead cells. The action-Tracker Red-555 can dye the cell microfilaments into Red without separating the species, and the cell adhesion state and proliferation depth of the cells can be observed under laser confocal when the cells in the stent are dyed simultaneously with DAPI (figure 7).
And the comprehensive result analysis shows that the cat adipose mesenchymal stem cells have good adhesion and proliferation states in the composite scaffold.
5. Expression of anti-inflammatory factors related to cell supernatant cultured by composite scaffold
TSG-6 is a multifunctional protein whose expression is up-regulated in the presence of inflammation in tissues. It has been found that TGF-beta is a transforming growth factor with anti-fibrotic activity in acute liver injury (Wang, S, lee, J S and Hyun, J, et al, tumor necrosis factor-induced gene 6promotes liver regeneration in mice with acute liver injury.Stem Cell Res Ther,2015,6:20), a key regulator of physiological and pathological fibrosis (Santibanez, J F, quintanila, M and Bernabeu, C.TGF-beta/TGF-beta receptor system and its role in physiological and pathological conditions.Clin Sci (Lond), 2011,121 (6): 233-251.), which is conductive to all stages of liver disease progression, from initial liver injury to inflammation and fibrosis, to cirrhosis and cancer (Fabregat, I, moreno-Cares, J and Sanchez, A, et al, TGF-beta signalling and liver disease.FEBS, 2016,283 (12): 2219-2232).
EGF, an EGF, has similar actions to the two factors above, and can promote cell growth, proliferation, survival, differentiation, etc., and can normally promote physiological processes such as wound healing, organ regeneration, etc., but excessive activation causes cancer (Guo Xiaojiang. EGF finder: stan Like J. Nature, year, 43 (04): 308-312). By combining the above background researches on three factors, we performed detection of three anti-inflammatory factors on three-dimensional culture cell supernatants, as follows:
(1) Taking cat adipose mesenchymal stem cells of P5 generation to count 1x10 6 Paving the composite bracket in a 6-hole plate, putting 4 brackets in each hole, and inoculating 500 mu L of cell suspension;
(2) After 3 days of cell growth on the scaffolds, culture supernatants were collected and assayed using ELISA kits for Cat-TSG-6, cat-TGF-beta and Cat-EGF, respectively.
The detection results show (FIG. 8) that the expression levels of TSG-6, TGF-beta and EGF in the composite scaffold supernatant were significantly increased.
Claims (9)
1. A composite scaffold for three-dimensional culture of stem cells, characterized in that: the preparation method of the composite scaffold is characterized in that the acellular apple fruits are taken as a framework, and the acellular apple fruits are crosslinked with a pig liver acellular matrix to form the composite scaffold, and the preparation method comprises the following steps:
(1) Sequentially carrying out cell removal treatment on the pork liver by using EDTA, SDS and DNase I enzymes, freeze-drying and crushing the treated pork liver to obtain a pork liver cell removal matrix;
(2) Cutting apples into pieces, performing cell removal treatment by SDS (sodium dodecyl sulfate), and freeze-drying to obtain a framework;
(3) Adding the pig liver acellular matrix and pepsin into a dilute acid solution, stirring to form a uniform solution, regulating the pH of the solution to 7-8, dialyzing to prepare a pig liver acellular matrix solution with the concentration of 2-9mg/mL, adding the acellular treated apple fruits, simultaneously adding EDC and NHS, soaking at room temperature, performing ultrasonic treatment, and cleaning to obtain the pig liver acellular matrix.
2. The composite stent of claim 1, wherein: in step (3), the pH of the solution was 7.4.
3. The composite stent of claim 1, wherein: in the step (3), the concentration of the pig liver acellular matrix solution is 5mg/mL.
4. The composite stent of claim 1, wherein: in the step (3), the molar ratio of EDC to NHS is 1:1.
5. the composite stent of claim 1, wherein: in the step (3), the ultrasonic frequency is 50-200KHZ, and the time is 10-60min.
6. The composite stent of claim 1, wherein: the concentration of EDTA, SDS and DNase I type enzyme is 0.02%, 5% and 0.01% respectively.
7. Use of the composite scaffold of claim 1 in three-dimensional culture of stem cells.
8. The use according to claim 7, wherein: the stem cells are adipose mesenchymal stem cells.
9. The use according to claim 7, wherein: the composite scaffold is favorable for adhesion and proliferation of stem cells and promotes expression of TSG-6, TGF-beta and EGF cytokines.
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