CN115737929A - Preparation method of self-assembled degradable tissue engineering membrane capable of obtaining monolayer cells - Google Patents
Preparation method of self-assembled degradable tissue engineering membrane capable of obtaining monolayer cells Download PDFInfo
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Abstract
The invention discloses a preparation method of a self-assembled degradable tissue engineering membrane capable of obtaining monolayer cells and a method for culturing monolayer epithelial cells on the self-assembled membrane and separating the monolayer cells. The invention takes Chitosan (CS) and Sodium Alginate (SA) as main raw materials, and prepares the deformable tissue engineering membrane by a self-assembly and ion replacement method, and the deformable tissue engineering membrane can pass Ca 2+ And Na + The alternating process achieves controlled deformation. Culturing the cells by using the membrane in a relaxation state until the cells on the surface of the membrane form a compact monolayer, and then passing through CaCl 2 And (3) treating the solution to enable the membrane to shrink rapidly so as to separate the monolayer cells on the membrane from the lower self-assembled membrane. The invention has the advantages that the material is low in cost and non-toxic, has good biocompatibility, biodegradability and film-forming property, can provide better environmental conditions for the growth of cells, and is beneficial to cell proliferation.
Description
Technical Field
The invention relates to the technical field of pharmaceutical preparations, in particular to a preparation method of a self-assembled degradable tissue engineering membrane capable of obtaining monolayer cells.
Background
Chitosan (Chitosan, CS) is a dephthaloyl product of chitin, a naturally biodegradable polycationic polysaccharide. The chitosan has the advantages of no toxicity, good biocompatibility, biodegradability and the like, and is widely applied in biomedicine, pharmacy and the like. CS is rich in amino and hydroxyl groups within the molecule, which can form hydrogen bonds, and thus CS is insoluble in water and organic solvents, but soluble in (dilute) acid solutions.
Sodium Alginate (SA) is an anionic polysaccharide extracted from marine brown algae, and mainly comprises 1-4 linked alpha-L-mannuronic acid and beta-D-mannuronic acid residues. Sodium alginate has been widely used and studied due to its biocompatibility, biodegradability, low cost, non-toxicity and good film-forming properties. However, the large number of free hydroxyl groups (-OH) and carboxylates (-COO-) distributed along the main chain of sodium alginate results in high hydrophilicity of sodium alginate, making sodium alginate exhibit poor mechanical properties and thermal stability due to its rigidity and brittleness. Therefore, there is a need to interact sodium alginate with other materials to improve water sensitivity and mechanical properties.
The amino group makes CS have positive charge, while under neutral and weakly alkaline conditions, the carboxyl group makes SA have a large negative charge, which is the basis for CS and SA to be capable of electrostatic adsorption self-assembly. Layer-by-layer self-assembly is a technology for effectively improving the mechanical property of the film, and the electrostatic action among ions is used as the driving force for film formation. With this method, the structure and thickness of the self-assembled membrane can be controlled, and the biofunctional macromolecules can be easily introduced into the membrane due to the non-specificity of electrostatic interactions.
The layer-by-layer self-assembled CS/SA film is water-soluble when Ca is used 2+ SA in the CS/SA film is converted into water-insoluble Calcium Alginate (CA), so that the film sheet can keep a stable shape in water. The CS/CA membrane shrinks significantly compared to the previous CS/SA membrane. But if the CS/CA membrane is re-soaked by NaCl solution with a certain concentration, part of Ca in the membrane 2+ Then can be covered by Na + And replaced again, resulting in the diaphragm expanding and becoming voluminous. The volume of the chitosan/sodium alginate/calcium alginate self-assembled membrane has obvious responsiveness to the concentration of Na + and Ca < 2+ > ions.
Therefore, it is an urgent technical problem to be solved by those skilled in the art to provide a preparation method for obtaining a tissue engineering in vitro monolayer cell self-assembled degradable tissue engineering membrane.
Disclosure of Invention
In view of this, the invention provides
In order to achieve the purpose, the invention adopts the following technical scheme:
a preparation method of a self-assembled degradable tissue engineering membrane capable of obtaining monolayer cells comprises the following steps:
(1) Under magnetic stirring, dissolving sodium alginate, glycerol and gelatin in water, and continuously stirring to obtain sodium alginate/glycerol/gelatin solution for later use;
(2) Dissolving chitosan, sodium chloride, glycerol and gelatin in HAc solution, and stirring at room temperature to obtain chitosan/NaCl 2 Glycerin/gelatin solution for use;
(3) Putting the glass plate into a sodium alginate/glycerol/gelatin solution for soaking, taking out, putting the glass plate into a biochemical incubator, and drying to obtain a glass slide with a sodium alginate film;
(4) Soaking the glass slide with the sodium alginate film in a chitosan/NaCl/glycerol/gelatin solution, taking out and putting the glass slide into a biochemical incubator for drying;
(5) Repeating the step (3) and the step (4) until the designed layer number is reached, and obtaining the glass slide with the layer-by-layer self-assembly film;
(6) Immersing glass slide with layer-by-layer self-assembled membrane into CaCl 2 Taking out the solution, drying the solution at room temperature, and immersing the dried solution into ethanol;
(7) And taking the membrane from the glass plate, drying and storing to obtain the self-assembled degradable tissue engineering membrane capable of obtaining the monolayer cells.
Further, the preparation method of the sodium alginate/glycerol/gelatin solution in the step (1) comprises the steps of dissolving 1.0-2.0g of sodium alginate, 0.5-2mL of glycerol and 0.5-2mL of gelatin in 100mL of water, and continuously stirring for 2-6h at 100-200 r/min to obtain a sodium alginate/glycerol/gelatin solution;
wherein the gelatin concentration is 0.05-0.2g/mL.
The beneficial effect of adopting the further scheme is that: the method can prepare the macromolecular monolayer membrane containing sodium alginate in the layer-by-layer self-assembled tissue engineering membrane.
Further, the preparation method of the chitosan/NaCl/glycerol/gelatin solution in the step (2) comprises the following steps: dissolving 0.5-2.0g of chitosan, 0.5-1.5g of sodium chloride, 0.5-2mL of glycerol and 0.05-0.2mL of gelatin in 100mL of HAc solution, and stirring at room temperature at 100-200 rpm for 2-6h;
wherein the gelatin concentration is 0.05-0.2g/mL, and the HAc solution concentration is 0.5-2 (v/v).
The beneficial effect of adopting the further scheme is that: preparing a macromolecular monolayer membrane containing chitosan in the layer-by-layer self-assembled tissue engineering membrane
Further, the dipping time in the step (3) is 5-20min; the drying temperature is 30-40 ℃, and the drying time is 0.5-3h.
Furthermore, in the step (4), the dipping time is 5-20min, the drying temperature is 30-40 ℃, and the drying time is 0.5-3h.
The beneficial effect of adopting the further scheme is that: the upper polymer membrane is shaped and then overlapped with a new polymer layer, so that the structure uniformity of each membrane can be ensured.
Further, the number of the design layers in the step (5) is 2-8.
Further, the CaCl in the step (6) 2 The solution concentration is 0.5-2.5% (w/w), and the soaking time is 15-40min;
the ethanol concentration is 50-80% (v/v), and the soaking time is 2-8min.
The beneficial effect of adopting the further scheme is that: using CaCl 2 Promote the sodium alginate in the polymer membrane to be converted into calcium alginate, and improve the stability of the membrane in the water solution.
The invention also provides a method for culturing the monolayer epithelial cells and separating the monolayer cells on the self-assembled degradable tissue engineering membrane, which comprises the following steps:
1) After the self-assembled degradable tissue engineering membrane is subjected to moist heat sterilization treatment, placing the membrane into sterile distilled water for later use;
2) Selecting normal growth bovine kidney epithelial cells for passage treatment, treating for 48h, taking the cells with anchorage rate of more than 80% as cells for experiment, digesting the cells, and adjusting cell concentration to 1-2 x 10 5 Per mL;
3) Placing the patches in a 48-well plate or a 96-well plate, adding 200. Mu.L of bovine kidney epithelial cell suspension to each patch, and placing the patches in a medium containing 5% CO 2 Culturing in a 37 ℃ incubator by using a DMEM medium;
4) Cells adhere to the wall on the membrane and grow, and the culture solution is replaced every 24h in the period;
5) When the cell fusion rate reaches more than 95%, removing the original DMEM medium, and cleaning each membrane by using PBS buffer solution;
6) Adding 200 μ L of 0.5-5.0% sterile NaCl solution into the culture well containing the membrane, incubating for 5-20min, and washing with DMEM medium for 2-4 times;
7) Adding 200 μ L of 0.2-0.8% sterile CaCl into culture well containing membrane 2 Incubating for 5-20min;
8) Observing the membrane and the cells under the mirror, and after the membrane shrinks and the monolayer cells are separated from the membrane, discarding CaCl 2 And (4) incubating the solution with a DMEM medium again to culture the epithelial cells of the monolayer and separating the cells of the monolayer.
Further, the wet heat sterilization treatment method is sterilization at 121 ℃ and 0.10MPa for 20min.
The beneficial effect of adopting the above-mentioned further scheme lies in: by NaCl and CaCl 2 The expansion and contraction of the diaphragm volume are influenced, the separation of the monolayer cells and the diaphragm is promoted, and finally the monolayer epithelial cells are obtained.
The invention has the beneficial effects that: the invention takes Chitosan (CS) and Sodium Alginate (SA) as main raw materials, prepares a deformable tissue engineering membrane by a self-assembly and ion replacement method, and can pass Ca 2+ And Na + Control by alternative treatmentAnd (5) deformation forming. Culturing the cells by using the membrane in a relaxation state until the cells on the surface of the membrane form a compact monolayer, and then passing through CaCl 2 And (3) treating the solution to enable the membrane to shrink rapidly, so that the monolayer cells on the membrane are separated from the lower self-assembled membrane. Therefore, the invention can obtain monolayer cells for tissue engineering in an in vitro state through the layer-by-layer self-assembled membrane sheet with the deformability.
Drawings
FIG. 1 is a schematic diagram of a self-assembled degradable tissue engineering membrane provided by the present invention;
FIG. 2 is a flow chart of the preparation of the self-assembled degradable tissue engineering membrane provided by the present invention;
FIG. 3 is a line graph of expansion and contraction performance of the layer-by-layer self-assembled tissue engineering membrane provided by the invention;
FIG. 4 is a graph of the expansion and contraction over time provided by the present invention;
FIG. 5 is a schematic diagram of the swelling ratio of the CA membrane, the CS membrane and the layer-by-layer self-assembled tissue engineering membrane provided by the invention;
FIG. 6 is a water vapor permeability diagram of the CA membrane, the CS membrane and the layer-by-layer self-assembled tissue engineering membrane provided by the invention;
FIG. 7 is a fluorescence image of AM/PI stained renal epithelial cells provided by the present invention;
FIG. 8 is a graph of activity of kidney epithelial cells measured at days 1, 3, and 5 of CCK-8 provided by the present invention;
FIG. 9 is a graph showing the results of PCNA measurements on different groups of renal epithelial cells at different times according to the present invention;
FIG. 10 is a graph of the determination of β -Catenin in renal epithelial cells in different time groups according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
Example 1
A preparation method of a self-assembled degradable tissue engineering membrane capable of obtaining monolayer cells comprises the following steps:
(1) Under magnetic stirring, 1.5g of sodium alginate, 1mL of glycerol and 1mL (0.1 g/mL) of gelatin are dissolved in 100mL of water, and stirring is continuously carried out for 4 hours at 150 revolutions/min to obtain a sodium alginate/glycerol/gelatin solution for later use;
(2) Dissolving 1.5g chitosan, 1g sodium chloride, 1mL glycerol and 0.1mL gelatin in 100mL HAc solution, and stirring at room temperature 150 rpm for 4h to obtain chitosan/NaCl 2 Glycerin/gelatin solution for use;
(3) Soaking the glass plate in sodium alginate/glycerol/gelatin solution for 10min, taking out, and oven drying in a biochemical incubator at 37 deg.C for 2 hr to obtain glass slide with sodium alginate membrane;
(4) Immersing glass slide with sodium alginate film into chitosan/CaCl 2 Soaking in glycerol/gelatin solution for 10min, taking out, and oven drying in biochemical incubator for 2 hr;
(5) Repeating the step (3) and the step (4) until the designed number of layers is 6, and obtaining the glass slide with the layer-by-layer self-assembly film;
(6) 1.5% (w/w) CaCl impregnated with slide glass with layer-by-layer self-assembled film 2 Soaking in the solution for 30min, drying at room temperature, and soaking in 75% (v/v) ethanol for 5min;
(7) And taking the membrane from the glass plate, drying and storing to obtain the self-assembled degradable tissue engineering membrane capable of obtaining monolayer cells.
1.5% of CaCl 2 After treatment, the self-assembled degradable tissue engineering membrane (LBLSM) is insoluble in water due to the conversion of Sodium Alginate (SA) to Calcium Alginate (CA). Whereas in the subsequent 5% NaCl treatment, slight expansion of LBLSM occurred. NaCl treatment for 4h and CaCl 2 Treatment for 12h promoted the maximum expansion/contraction ratio of LBLSM. In the first 4 treatment cycles, the membrane surface was expanded to 120% and 110% of the initial area, respectively, with NaCl for 4h, and CaCl 2 The film surface shrinks when treated for 12h80% (FIG. 3). Furthermore, the initial network structure of LBLSM is relatively loose and therefore in Na + In the first 4 cycles of the replacement, more Ca 2+ The site is replaced. Na (Na) + After treatment, the network structure of LBLSM tends to be dense and stable, the surface area tends to be stable, returning only to the original surface area. But since the design of the membrane is updated daily in clinical treatments, the deformation of the first 4 cycles should be of more concern. FIG. 4 shows 5% NaCl and 1.5% CaCl 2 The change in membrane surface area after the cycling treatment was consistent with figure 3. In addition, LBLSM has more excellent water absorption capacity than CA and CS membranes, and the water vapor transmission capacity is the same (fig. 5-6), which indicates that LBLSM not only can better absorb wound exudate, maintain wound moist environment, but also can promote evaporation of excess moisture and interstitial fluid, accelerate wound scabbing, and maintain wound sterile condition.
Renal epithelial cells were selected to evaluate the biocompatibility of LBLSM and the effect of membrane deformation on cell growth in vitro. AM/PI staining (FIG. 7) and CCK-8 assay results (FIG. 8) showed that LBLSM (undeformed) treated cells were more green fluorescent and grew faster than the control, indicating that LBLSM by itself significantly promoted cell growth. Most of the epithelial cells are spindle-shaped and are very suitable for spreading and growing on the oriented surface of the material, so that the high hydrophilicity, high orientation and proper surface roughness of the LBLSM can provide better environmental conditions for the growth of the epithelial cells. Simultaneously, through NaCl/CaCl 2 After the regular deformation treatment, the cells on the LBLSM have higher density, higher growth speed and higher cell fusion degree than other groups of cells, which indicates that the regular mechanical deformation is favorable for cell division and proliferation. Proliferating Cell Nuclear Antigen (PCNA) is closely associated with cellular DNA synthesis and plays an important role in cell proliferation. In addition, beta-catenin is a multifunctional protein widely existing in various types of cells and is also important for cell proliferation, differentiation and apoptosis. From the results of FIGS. 9 and 10, LBLSM group and LBLSM + NaCl + CaCl 2 The expression of the PCNA and the beta-Catenin groups are stronger than those of the control group, and LBLSM + NaCl + CaCl 2 The highest expression of the PCNA and β -Catenin groups indicates that mechanical deformation of LBLSM favors cell proliferation.
Example 2
A preparation method of a self-assembled degradable tissue engineering membrane capable of obtaining monolayer cells comprises the following steps:
(1) Under magnetic stirring, 1.0g of sodium alginate, 2mL of glycerol and 0.5mL of gelatin are dissolved in 100mL of water, and the mixture is continuously stirred for 2 hours at 200 revolutions per minute to obtain a sodium alginate/glycerol/gelatin solution for later use;
(2) Dissolving 0.5g chitosan, 1.5g sodium chloride, 0.5mL glycerol and 0.2mL gelatin in 100mL HAc solution, and stirring at room temperature for 2h at 200 rpm to obtain chitosan/NaCl 2 Glycerin/gelatin solution for use;
(3) Soaking the glass plate in sodium alginate/glycerol/gelatin solution for 20min, taking out, and oven drying in a biochemical incubator at 30 deg.C for 3h to obtain glass slide with sodium alginate membrane;
(4) Soaking the glass slide with the sodium alginate membrane in a chitosan/NaCl/glycerol/gelatin solution for 20min, taking out and putting the glass slide into a biochemical incubator to dry for 3h at 30 ℃;
(5) Repeating the step (3) and the step (4) until the designed layer number is 2, and obtaining the glass slide with the layer-by-layer self-assembly film;
(6) 2.5% by immersion of slide glass with layer-by-layer self-assembled film pieces in CaCl 2 Soaking in 50% ethanol for 8min after drying at room temperature for 15 min;
(7) And taking the membrane from the glass plate, drying and storing to obtain the self-assembled degradable tissue engineering membrane capable of obtaining the monolayer cells.
Example 3
A preparation method of a self-assembled degradable tissue engineering membrane capable of obtaining monolayer cells comprises the following steps:
(1) Under magnetic stirring, 2.0g of sodium alginate, 0.5mL of glycerol and 2mL of gelatin are dissolved in 100mL of water, and stirring is continuously carried out for 6 hours at 100 revolutions per minute to obtain a sodium alginate/glycerol/gelatin solution for later use;
(2) Dissolving 2.0g chitosan, 0.5g sodium chloride, 2mL glycerol and 0.05mL gelatin in 100mL HAc solution, and stirring at room temperature for 6h at 100 rpm to obtain chitosan/NaCl 2 Glycerin/gelatin solution for use;
(3) Soaking the glass plate in sodium alginate/glycerol/gelatin solution for 5min, taking out, and oven drying in a biochemical incubator at 40 deg.C for 0.5h to obtain glass slide with sodium alginate membrane;
(4) Soaking the glass slide with the sodium alginate film in a chitosan/NaCl/glycerol/gelatin solution for 5min, taking out and putting the glass slide into a biochemical incubator to dry for 0.5h at 40 ℃;
(5) Repeating the step (3) and the step (4) until the designed number of layers reaches 8, and obtaining the glass slide with the layer-by-layer self-assembly film;
(6) 0.5% by immersion of slide glass with layer-by-layer self-assembled film pieces in CaCl 2 Soaking in the solution for 40min, drying at room temperature, and soaking in 80% ethanol for 2min;
(7) And taking the membrane from the glass plate, drying and storing to obtain the self-assembled degradable tissue engineering membrane capable of obtaining the monolayer cells.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (9)
1. A preparation method of a self-assembled degradable tissue engineering membrane capable of obtaining monolayer cells is characterized by comprising the following steps:
(1) Under magnetic stirring, dissolving sodium alginate, glycerol and gelatin in water, and continuously stirring to obtain sodium alginate/glycerol/gelatin solution for later use;
(2) Dissolving chitosan, sodium chloride, glycerol and gelatin in HAc solution, and stirring at room temperature to obtain chitosan/NaCl 2 Glycerin/gelatin solution for use;
(3) Putting the glass plate into a sodium alginate/glycerol/gelatin solution for soaking, taking out, putting the glass plate into a biochemical incubator, and drying to obtain a glass slide with a sodium alginate film;
(4) Soaking the glass slide with the sodium alginate film in a chitosan/NaCl/glycerol/gelatin solution, taking out and putting the glass slide into a biochemical incubator for drying;
(5) Repeating the step (3) and the step (4) until the designed layer number is reached, and obtaining the glass slide with the layer-by-layer self-assembly film;
(6) Immersing glass slide with layer-by-layer self-assembled membrane into CaCl 2 Taking out the solution, drying the solution at room temperature, and immersing the dried solution into ethanol;
(7) And taking the membrane from the glass plate, drying and storing to obtain the self-assembled degradable tissue engineering membrane capable of obtaining the monolayer cells.
2. The method for preparing the self-assembled degradable tissue engineering membrane capable of obtaining the monolayer cells according to claim 1, wherein the sodium alginate/glycerol/gelatin solution in the step (1) is prepared by dissolving 1.0-2.0g of sodium alginate, 0.5-2mL of glycerol and 0.5-2mL of gelatin in 100mL of water, and continuously stirring for 2-6h at 100-200 rpm to obtain a sodium alginate/glycerol/gelatin solution;
wherein the gelatin concentration is 0.05-0.2g/mL.
3. The method for preparing the self-assembled degradable tissue engineering membrane capable of obtaining the monolayer of cells according to claim 1, wherein the method for preparing the chitosan/NaCl/glycerol/gelatin solution in the step (2) comprises the following steps: dissolving 0.5-2.0g of chitosan, 0.5-1.5g of sodium chloride, 0.5-2mL of glycerol and 0.05-0.2mL of gelatin in 100mL of HAc solution, and stirring at room temperature at 100-200 rpm for 2-6h;
wherein the gelatin concentration is 0.05-0.2g/mL, and the HAc solution concentration is 0.5-2 (v/v).
4. The method for preparing the self-assembled degradable tissue engineering membrane capable of obtaining the monolayer cells according to claim 1, wherein the soaking time in the step (3) is 5-20min; the drying temperature is 30-40 ℃, and the drying time is 0.5-3h.
5. The method for preparing the self-assembled degradable tissue engineering membrane capable of obtaining the monolayer cells according to claim 1, wherein the soaking time in the step (4) is 5-20min, the drying temperature is 30-40 ℃, and the drying time is 0.5-3h.
6. The method for preparing a self-assembled degradable tissue engineering membrane capable of obtaining monolayer cells according to claim 1, wherein the designed number of layers in step (5) is 2-8.
7. The method for preparing the self-assembled degradable tissue engineering membrane capable of obtaining the monolayer of cells as claimed in claim 1, wherein the CaCl in the step (6) 2 The solution concentration is 0.5-2.5% (w/w), and the soaking time is 15-40min;
the ethanol concentration is 50-80% (v/v), and the soaking time is 2-8min.
8. A method for culturing a monolayer of epithelial cells and isolating the monolayer of cells on the self-assembled degradable tissue engineering sheet according to any one of claims 1 to 7, comprising the steps of:
1) After the self-assembled degradable tissue engineering membrane is subjected to moist heat sterilization treatment, placing the membrane into sterile distilled water for later use;
2) Selecting normal growth bovine kidney epithelial cells for passage treatment, treating for 48h, taking the cells with anchorage rate of more than 80% as cells for experiment, digesting the cells, and adjusting cell concentration to 1-2 x 10 5 Per mL;
3) Placing the patches in a 48-well plate or a 96-well plate, adding 200. Mu.L of bovine kidney epithelial cell suspension to each patch, and placing the patches in a medium containing 5% CO 2 Culturing in a 37 ℃ incubator by using a DMEM medium;
4) Cells adhere to the wall on the membrane and grow, and the culture solution is replaced every 24 hours;
5) When the cell fusion rate reaches more than 95%, removing the original DMEM medium, and cleaning each membrane by using PBS buffer solution;
6) Adding 200 μ L of 0.5-5.0% sterile NaCl solution into the culture well containing the membrane, incubating for 5-20min, and washing with DMEM medium for 2-4 times;
7) Adding 200 μ L of 0.2-0.8% sterile CaCl into culture well containing membrane 2 Incubating for 5-20min;
8) Observing the membrane and the cells under the mirror, and after the membrane shrinks and the monolayer cells are separated from the membrane, discarding CaCl 2 And (4) incubating the solution with a DMEM medium again to culture the epithelial cells of the monolayer and separating the cells of the monolayer.
9. The method for culturing the monolayer of epithelial cells on the self-assembled degradable tissue engineering membrane and separating the monolayer of cells as claimed in claim 8, wherein the moist heat sterilization treatment is performed at 121 ℃ and 0.10MPa for 20min.
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| US20050233442A1 (en) * | 2004-04-08 | 2005-10-20 | Fuji Photo Film Co., Ltd. | Carrier for cell culture |
| CN106924817A (en) * | 2017-03-02 | 2017-07-07 | 浙江大学 | A kind of ultra-thin carrier cell piece and preparation method thereof |
| CN115678828A (en) * | 2022-10-12 | 2023-02-03 | 青岛申康干细胞生物科技有限公司 | Preparation method and application of layer-by-layer self-assembled tissue engineering membrane |
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| US20050233442A1 (en) * | 2004-04-08 | 2005-10-20 | Fuji Photo Film Co., Ltd. | Carrier for cell culture |
| CN106924817A (en) * | 2017-03-02 | 2017-07-07 | 浙江大学 | A kind of ultra-thin carrier cell piece and preparation method thereof |
| CN115678828A (en) * | 2022-10-12 | 2023-02-03 | 青岛申康干细胞生物科技有限公司 | Preparation method and application of layer-by-layer self-assembled tissue engineering membrane |
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