CN115232787B - Preparation method of extracellular body based on acellular matrix three-dimensional scaffold culture - Google Patents
Preparation method of extracellular body based on acellular matrix three-dimensional scaffold culture Download PDFInfo
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Abstract
The invention discloses a preparation method of an extracellular body based on decellularized matrix three-dimensional scaffold culture, and belongs to the field of tissue engineering regenerative medicine. The preparation method of the exosome provided by the invention comprises the following steps: and inoculating the mesenchymal stem cells on a acellular matrix three-dimensional scaffold, culturing, and separating to obtain exosomes. The invention uses acellular matrix as raw material, constructs bionic natural cell growth microenvironment from two aspects of material composition and space structure, can further optimize the biological function of exosomes, and fully plays the therapeutic potential of exosomes.
Description
Technical Field
The invention relates to the field of tissue engineering regenerative medicine, in particular to a preparation method of an extracellular body based on decellularized matrix three-dimensional scaffold culture.
Background
Mesenchymal stem cells (MSCs, mesenchymal stem cells) are important members of the stem cell family, originate from early-stage mesoderm, belong to multipotent stem cells, and are originally found in bone marrow, and are increasingly attracting attention due to their multipotent differentiation potential, hematopoietic support, promotion of stem cell implantation, immune regulation, self-replication and the like.
For example, the mesenchymal stem cells can be differentiated into various tissue cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelial and the like under specific induction conditions in vivo or in vitro, and still have multidirectional differentiation potential after continuous subculture and cryopreservation, and can be used as ideal seed cells for repairing tissue organ injury caused by aging and pathological changes.
Because mesenchymal stem cells have multidirectional differentiation potential, students in the field of regenerative medicine hold great expectations for repairing damaged tissues or organs by using MSCs, and the safety of the MSCs is a major concern for scientists at present and is also a major obstacle for clinical application of the MSCs. With the increasing depth of research, researchers have realized that the therapeutic effects of MSCs are largely dependent on paracrine action to secrete a variety of active factors. Among these paracrine factors, exosomes (exomes, exo) are considered as the most biologically effective substances. Compared with MSCs, exo has the advantages of easy extraction, low immunogenicity, no tumorigenicity, low transplantation risk, low transportation and preservation requirements and the like, so that Exo is considered to be an ideal substitute for MSCs. The traditional MSCs-Exo is mostly obtained through two-dimensional plane culture extraction, cells grow in a three-dimensional state in vivo, the two-dimensional plane culture cannot well replicate the growth microenvironment of the cells in a real state, the cell performance in the three-dimensional bracket culture is obviously different from that in the two-dimensional plane culture, and the cell-cell and cell-matrix interaction in the three-dimensional culture can better simulate the growth microenvironment of the cells. In addition to three dimensional results, the composition of the scaffold material is also an important component of the cell growth microenvironment.
Disclosure of Invention
The invention provides a preparation method of an exosome based on acellular matrix three-dimensional scaffold culture, which adopts acellular matrix three-dimensional scaffold culture cells to prepare the exosome, optimizes the biological functions of the exosome and fully exerts the therapeutic potential of the exosome.
The invention firstly provides a preparation method of exosomes, which comprises the following steps: and inoculating the mesenchymal stem cells on a acellular matrix three-dimensional scaffold, culturing, and separating to obtain exosomes.
In the preparation method, the acellular matrix three-dimensional scaffold is prepared from acellular matrix;
the acellular matrix is prepared by a method comprising the following steps:
(1) Carrying out decellularization treatment on animal tissues to obtain decellularized matrix homogenate;
(2) Adding acetic acid solution into the acellular matrix homogenate, stirring, and volatilizing part of acetic acid to obtain the acellular matrix.
The animal tissue is any one of cartilage tissue, meniscus tissue, bone tissue, tendon, muscle, ligament and skin;
the decellularized treatment method of the animal tissue is a physical method, a chemical method or an enzymatic method.
In the step (2), the mass percentage concentration of the acetic acid solution is 20% -100%; specifically, the content of the active ingredients is 80% -100%.
The acetic acid solution is continuously added into the acellular matrix homogenate in a dropwise manner until the acellular matrix homogenate is changed from milky to transparent.
In the step (2), the stirring temperature is 0-8 ℃ and the stirring time is 12-72 h;
the stirring is carried out in a closed container;
the acetic acid volatilizing method is that acetic acid is volatilized by magnetic stirring under an exhaust fan;
the mass percentage concentration of the acellular matrix is 5-10%.
In the preparation method, the acellular matrix three-dimensional scaffold is obtained by taking the acellular matrix as printing ink through low-temperature deposition 3D printing or by adopting a freeze drying method.
The low temperature range is-10 to-80 ℃; specifically, the temperature can be-20 ℃;
in the preparation method, the culture is carried out for 48-96 hours by adopting a culture solution containing serum or Ultroser G, then the serum culture solution without exosomes is replaced for continuous culture for 48-96 hours, and the culture supernatant is collected for separation treatment, thus obtaining the exosomes.
Specifically, in the culture solution containing the serum, the volume concentration of the serum in the culture solution is 10%; the serum is fetal bovine serum; the culture solution is DMEM/F-12 complete culture solution.
The invention also provides an exosome prepared by the preparation method.
The invention has the following beneficial effects:
the change of the micro-environment of the cells has great influence on the functions of exosomes, so the biological functions of the exosomes can be further optimized by adjusting the growth environment of the cells; the invention uses acellular matrix as raw material, constructs bionic natural cell growth microenvironment (acellular matrix three-dimensional scaffold) from two aspects of material composition and space structure, can further optimize the biological function of exosomes, and fully plays the therapeutic potential of exosomes.
Drawings
FIG. 1 is a decellularized cartilage matrix prepared in example 1;
FIG. 2 is a plot of viscosity versus shear rate of the acellular cartilage matrix prepared in example 1;
FIG. 3 shows cell morphology in flasks (conventional planar two-dimensional culture) and decellularized matrix three-dimensional scaffolds;
FIG. 4 shows the morphology of the extracellular matrix prepared by flask culture and decellularized matrix three-dimensional scaffold culture;
FIG. 5 shows the migration capacity of stem cells by extracellular fluid prepared by flask culture and decellularized matrix three-dimensional scaffold culture;
FIG. 6 shows the promotion of macrophage polarization to M2 type by extracellular fluid prepared by flask culture and decellularized matrix three-dimensional scaffold culture.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof.
The experimental methods in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Dnase and Rnase used in the examples below were Dnase and Rnase, respectively, purchased from Sigma in the united states; product numbers were Dnase 10104159001, respectively; rnase 10109134001.
The phosphate buffer (PBS buffer) used in the following examples was prepared as follows: 8.0g NaCl, 0.2g KCl and 1.44g Na are weighed 2 HPO 4 、0.24g KH 2 PO 4 Dissolving in 800mL of distilled water, regulating the pH of the solution to 7.4 by using HCl, and finally adding distilled water to a volume of 1L to obtain the 0.01M PBS buffer solution.
Example 1 preparation of acellular matrix scaffolds
(1) Decellularization treatment of cartilage matrix
On an experimental animal operating table, a fresh pig knee joint is incised under a sterile condition, and cartilage tissues of a femur condyle, a patella and a tibia platform are taken after the pig knee joint is washed clean by sterile normal saline. The collected cartilage pieces are placed in a sterile wide-mouth bottle, sterile deionized water (the volume ratio of deionized water to cartilage tissue is 5:1) is added, and the cartilage pieces are subjected to 10 cycles of freezing and thawing (the freezing temperature is-80 ℃ C., and the thawing temperature is room temperature), and liquid is replaced once each cycle. Then the cartilage tissue is transferred into a tissue homogenizer, and is crushed intermittently at low temperature (the crushed cartilage tissue is put into a refrigerator with the temperature of minus 20 ℃ for 1 to 2 minutes after being crushed for 30 to 60 seconds) to obtain cartilage tissue homogenate. Adding trypsin-EDTA with the mass percentage concentration of 0.25%, wherein the volume ratio of the trypsin solution to the cartilage tissue homogenate is 1:5; treating on a shaking table at 37deg.C for 24 hr, rinsing with phosphate buffer solution (phosphate buffer saline, PBS), centrifuging at 10000rpm for 30min, pouring supernatant, adding nuclease solution with concentration of 50U/mL DNase and 1U/mL RNase into the precipitate, stirring thoroughly to remove nucleic acid substances, rinsing with sterile deionized water repeatedly for three days to remove residual reagent, centrifuging at 10000rpm for 60min to obtain precipitate which is acellular cartilage matrix homogenate, wherein the homogenate is opaque milky white
(2) Acid treatment of decellularized cartilage matrix homogenate
Acetic acid with the mass percentage concentration of 80% is continuously added into the acellular cartilage matrix homogenate until the matrix is completely dissolved (the matrix is milky white before being dissolved and semitransparent after being dissolved according to the judgment of the color), a bottle mouth is sealed by a sealing film at the temperature of 4 ℃ and magnetically stirred for 24 hours, so that the acellular cartilage matrix homogenate is fully dissolved, and the acellular cartilage matrix homogenate after being dissolved is transparent solution, but the concentration and the viscosity are low at the moment, and the 3D printability is still not achieved. Removing the sealing film, continuing magnetic stirring under an exhaust fan, accelerating volatilization of acetic acid, and obtaining a sticky state, wherein the 3D printability is achieved when the mass fraction is about 5-10%; the mass fraction of the acellular cartilage matrix obtained in this example is 6%, and the photograph is shown in fig. 1.
The mass fraction of the acellular cartilage matrix is determined by the following steps: firstly, measuring a certain amount of homogenized wet weight, then drying at 60 ℃ overnight, removing the solvent, and measuring the dry weight, namely the solute mass, and the dry weight-to-wet weight, namely the mass concentration.
The viscosity-shear rate curve of the acellular cartilage matrix is shown in fig. 2, and it can be seen from fig. 2 that the acellular cartilage matrix prepared by the present invention has shear thinning (test temperature is room temperature).
(3) Preparation of acellular matrix three-dimensional scaffold
Placing the acellular cartilage matrix prepared in the step (2) into a 3D printer (SUNP ALPHA-BP31, shangpu Boyuan (Beijing) biotechnology Co., ltd.) cylinder, extruding and printing by piston extrusion, and rapidly solidifying and molding the extruded acellular cartilage matrix on a freezing platform to obtain a bracket;
the print parameters were set as follows: the aperture is 500 mu m, the diameter of the spray head is 400 mu m, the thickness is 2mm, the fiber angle is 90 degrees, the push-out speed is 0.1mm/s, the printing speed is 5mm/s, the low-temperature freezing platform temperature is-20 ℃, the printing bin temperature is-20 ℃, and the printing spray head temperature is 4 ℃.
After printing, the scaffolds were placed in a freeze-dryer for sublimation drying under vacuum (< 100 mTorr) -60deg.C for 48h.
Placing the support after sublimation drying into a cross-linking agent for cross-linking, wherein the cross-linking temperature is 4 ℃, and the cross-linking time is 24 hours;
the cross-linking agent is a 95% (v/v) ethanol solution containing ethyl-dimethylamine-propyl carbodiimide (EDAC) and n-hydroxysuccinimide (NHS), wherein the concentration of EDAC is 50mmol/L; NHS concentration is 20mmol/L;
after crosslinking, soaking the mixture in PBS buffer solution for 2 hours, and removing redundant crosslinking agent; and after rinsing with the three distilled water, performing freeze drying sublimation treatment again (sublimation for 48 hours at the temperature of between 100mTorr and 60 ℃ under vacuum condition) to obtain the acellular matrix three-dimensional scaffold.
Example 2 preparation of extracellular exosomes
Bone marrow mesenchymal stem cells of SD rat were inoculated into culture flasks (10X 1 mm) 3 ) And the decellularized matrix three-dimensional scaffolds prepared in example 1, the number of cells inoculated was 100 tens of thousands, respectively, cultured in DMEM/F-12 complete medium (Gibco, usa, cat No. 8121643) having a concentration of 10% (volume fraction) of normal FBS (fetal bovine serum). 37 ℃,5% CO 2 After (carbon dioxide) incubation for 72h, the culture was aspirated and the cells were washed twice with PBS buffer for 1min each time. The whole culture broth was replaced with DMEM/F-12 with 10% (volume fraction) concentration of Exo serum (System Biosciences, cat. EXO-FBS-50A-1). 37 ℃,5% CO 2 (carbon dioxide) culturing for 72h, recovering cell supernatant into 50mL centrifuge tube under aseptic condition, centrifuging at 300 Xg for 10min, and discarding precipitate; then centrifuging at 2000 Xg for 10min, discarding the precipitate, and removing cells in the supernatant; centrifuging at 10000 Xg for 30min, removing cell debris from supernatant; next, the mixture was centrifuged at 100000 Xg for 120min, and the supernatant was discarded, and the precipitate contained Exo. The pellet was resuspended in PBS buffer (the volumes of PBS buffer and pellet were2-10:1, in this embodiment, specifically 5:1), centrifuging again at 100000×g for 120min, discarding supernatant, precipitating to obtain exosomes, and lyophilizing.
SD rat bone marrow mesenchymal stem cells (BMSCs) were inoculated into the decellularized matrix three-dimensional scaffolds (three-dimensional culture) and culture flasks (two-dimensional planar culture, 10X 1 mm) prepared in example 1 3 ) The cell quantity of inoculation is 100 ten thousand respectively, after the equal cells are fully adhered, DMEM/F12 cell culture solution containing 10% fetal bovine serum by volume concentration is added, the temperature is 37 ℃, and CO is 5% 2 The materials were obtained after culturing in a (carbon dioxide) incubator for 7 days, rinsed 2 times with PBS buffer, stained with a dead/alive fluorescent dye (thermo-filter, md. MP 03224, USA) for 20min, rinsed 2 times with PBS solution, and observed with confocal microscopy. The parameters associated with excitation of green and red fluorescence are set as follows: excitation wavelength: 535nm and 355nm, emission wavelength: 585nm and 460nm. The results are shown in FIG. 3. As can be seen from fig. 3, the two-dimensional planar cultured huchmscs grow in a spreading shape, obvious filiform actin is seen, cells are randomly arranged and distributed, contact with surrounding cells is less, the three-dimensional cultured huchmscs grow in a three-dimensional shape along the pores of the scaffold, and more mutual contact and interaction with the surrounding cells and the scaffold exist, so that the spatial structure can obviously influence the growth behavior of the cells.
Exos samples were prepared using an Exosome-TEM-easy kit (U.S. 101Bio Inc., P130), 3 drops of Exos suspension (10. Mu.L per drop) prepared from the lyophilized exosomes described above by a kit (Exosome-TEM-easy kit) were dropped onto a clean sealing film, 3 Formvar carbon coated EM copper mesh was suspended on the drop with the coating facing the drop, and left to stand for 10min. The clean sealing film was dropped with 30. Mu.L of wash buffer, and the copper mesh was transferred to the wash buffer using a fine tweezer, and washed 2 times for 30 seconds each time. 10. Mu.L of EM solution was dropped on the clean sealing film, the copper mesh was transferred to the drop, and the mixture was allowed to stand for 10 minutes. The washing buffer was again used for 2 washes of 30s each. The copper mesh was transferred to clean filter paper with the coating facing up and air dried overnight at room temperature. The next day, exos on the copper mesh was observed using a Transmission Electron Microscope (TEM). The results are shown in FIG. 4. As can be seen from FIG. 4, the two-layer biofilm structures of 2D-Exo and 3D-Exo are clearly seen, which indicates that the exosomes prepared in this example have the structural characteristics of exosomes.
2D-exo and 3D-exo media were prepared in 25. Mu.g/mL with DMEM/F-12 medium having 1% FBS by volume, and added to a 6-well plate at about 5X 10 5 After 24 hours, the individual cells (BMSC) were scratched with a 200u gun head, the gun head was vertical, the widths of the two groups of cells were guaranteed to be similar, the cells were gently washed 3 times with PBS buffer, the scratched cells were removed, 3mL of 2D-exo and 3D-exo culture solution were added respectively, DMEM/F-12 culture solution (without exosomes) with 1% of FBS volume concentration was added to the control group, and the mixture was put into 37℃CO of 5% 2 Incubator, culture for 24 hours, observe cell migration ability by photographing. The results are shown in FIG. 5. As can be seen from fig. 5, both the two-dimensional culture and the three-dimensional culture Exo exhibited an effect of promoting cell migration compared to the control group, wherein the promoting effect of 3D-Exo was more remarkable, indicating that 3D-Exo was able to promote cell migration better.
2D-exo and 3D-exo media were prepared at 25. Mu.g/mL using DMEM/F-12 media with 1% FBS volume concentration, and mouse M0 macrophages (10 5 And 3mL of 2D-exo and 3D-exo culture solution are respectively added into a 12-well plate, DMEM/F-12 culture solution (without exosomes) with the volume concentration of FBS of 1% is added into a control group, cells are cultured for 24 hours, 1% BSA (bovine serum albumin) is added into each tube, 100 mu L of BSA is added into each tube, incubation is carried out on ice for 30min, fluorescent primary antibody is added into the mixture at a preset optimal concentration (APC-CD 86:2.5mg/L and PE-CD206:5 mg/L), incubation is carried out on ice for 15-20 min, PBS buffer is used for 3 times, a low-temperature centrifuge is used for 600 Xg centrifugation for 5min, and cell sediment is resuspended into 300-500 mu LPBS buffer for detection by a flow cytometer. The results are shown in FIG. 6. As can be seen from fig. 6, both the two-dimensional culture and the three-dimensional culture Exo showed promotion of macrophage to M2 type polarization compared to the control group, wherein the promotion effect of 3D-Exo was more remarkable, indicating that 3D-Exo could better promote M0 macrophage to M2 type polarization of mice.
Claims (4)
1. A method for preparing exosomes, comprising the steps of: inoculating mesenchymal stem cells on a acellular matrix three-dimensional scaffold, culturing, and separating to obtain exosomes;
the acellular matrix three-dimensional scaffold is obtained by taking the acellular matrix as printing ink through low-temperature deposition 3D printing; the low temperature range is-10 to-80 ℃; the method comprises the following specific steps: placing the acellular matrix in a 3D printer charging barrel, extruding and printing in a piston extrusion mode, and rapidly solidifying and forming the extruded acellular cartilage matrix on a freezing platform to obtain a bracket;
after printing, placing the bracket into a freeze dryer for sublimation drying;
placing the support after sublimation drying into a cross-linking agent for cross-linking;
the cross-linking agent is a 95% ethanol solution containing ethyl-dimethylamine-propyl carbodiimide and n-hydroxysuccinimide in percentage concentration by volume, wherein the concentration of the ethyl-dimethylamine-propyl carbodiimide is 50mmol/L; the concentration of the n-hydroxysuccinimide is 20mmol/L;
after crosslinking, soaking the mixture in PBS buffer solution for 2 hours, and removing redundant crosslinking agent; rinsing with distilled water, and freeze-drying and sublimating again to obtain the acellular matrix three-dimensional scaffold;
the acellular matrix is prepared by a method comprising the following steps:
(1) Carrying out decellularization treatment on animal tissues to obtain decellularized matrix homogenate; the animal tissue is cartilage tissue;
(2) Adding an acetic acid solution into the acellular matrix homogenate, stirring, and volatilizing part of acetic acid to obtain the acellular matrix;
the acetic acid solution is continuously added into the acellular matrix homogenate in a dropwise manner until the acellular matrix homogenate is changed from milky to transparent;
the temperature of stirring is 0-8 ℃ and the time is 12-72 h;
the acetic acid volatilizing method is that acetic acid is volatilized by magnetic stirring under an exhaust fan;
the mass percentage concentration of the acellular matrix is 5-10%.
2. The method of manufacturing according to claim 1, characterized in that: the decellularized treatment method of the animal tissue is a physical method, a chemical method or an enzymatic method.
3. The preparation method according to claim 1 or 2, characterized in that: in the step (2), the mass percentage concentration of the acetic acid solution is 20% -100%.
4. The preparation method according to claim 1 or 2, characterized in that: the culture is that a culture solution containing serum or Ultroser G is adopted for culture for 48-96 hours, then the serum culture solution without exosomes is replaced for continuous culture for 48-96 hours, and culture supernatant is collected for separation treatment, so that the exosomes are obtained.
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| Wenting Yu等.Higher yield and enhanced therapeutic effects of exosomes derived from MSCs in hydrogel-assisted 3D culture system for bone regeneration.Biomaterials Advances.2022,第133卷摘要、第1页右栏-第2页左栏第3段、第2页2.2-2.3、图1. * |
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