CN115645628A - Rapid harvesting method of cell sheet layer and 'colloid + cell sheet' composite film - Google Patents
Rapid harvesting method of cell sheet layer and 'colloid + cell sheet' composite film Download PDFInfo
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Images
Abstract
The invention discloses a rapid harvesting method of a cell sheet layer and a 'colloid + cell sheet' composite film, which comprises the steps of inoculating cells on the surface of a substrate, dripping a glue solution on the surface of the substrate after culturing to obtain the cell sheet layer, irradiating to ensure that the glue solution is subjected to photo-crosslinking, then dripping excessive metal ion solution for ion crosslinking, and removing the excessive solution to easily remove the film layer compounded with the cell sheet layer to obtain the 'colloid + cell sheet' composite film. According to the method, a glue material and a cell sheet layer can be fully compounded, hydrogel with good mechanical properties can be obtained through photo-crosslinking and ion crosslinking, the compounding level of a colloid and the cell sheet layer can be improved through the characteristic of photo-generated aldehyde groups of the hydrogel, the binding force of a colloid and cell sheet thin film and a substrate is greatly reduced in the ion crosslinking process, the cell sheet layer with a large area can be harvested and compounded with the hydrogel, and the obtained colloid and cell sheet thin film is expected to be used as a biological patch and applied to skin wound repair, hemostasis and complete repair and regeneration of cartilage defects.
Description
Technical Field
The invention relates to the preparation problems of cell sheet technology and tissue engineering scaffolds in the field of biomedicine, in particular to a method for harvesting a cell sheet and compounding water gel.
Background
In recent years, cell sheet technology has been widely used in the field of tissue engineering because of its retention of high density Cell tissue, natural ECM secreted by mature cells, and the like, and can be engineered [ Long t.the Effect of sensory Stem Cell Sheets on Structural allogration health of Critical Sized febrile Defects in rice 2014. Biomaterials; 35:2752-2759]. For example, cell sheet technology is considered to bring a potential solution to the reduction of scars and even to a perfect regeneration state by skin repair. Cheng et al rapidly prepared Adipose-Derived Stem cell (ASC) sheets by L-ascorbic acid 2-phosphate stimulation and applied the ASC sheets in the field of tissue regeneration and Wound Healing, research shows that ASC sheets have necessary secretion factors, can improve Skin Wound Healing, and can generate new Skin with excellent quality [ Yu J.cell show compounded of adobe-Derived Stem Cells modified Skin Wound with Reduced tissue Format.acta Biomaterial 2018;77:191-200]. However, the cell sheet obtained in the prior art has the defects of poor mechanical strength, difficulty in obtaining a large-area cell sheet layer and the like, so that the application prospect of the cell sheet in full-layer skin injury repair is limited.
Based on this, it is considered that Hydrogel materials having good mechanical properties are characterized by both excellent biocompatibility and biodegradability [ Ghobril C.the Chemistry and Engineering of Polymeric Hydrogel Adhesives for Wund cleansing: a Tutorial.chemical Society Reviews 2015; 44-1820-1835 ], if the hydrogel with good mechanical strength is used for harvesting the cell sheet and the cell sheet with high bioactivity is compounded with the hydrogel, it is possible to harvest the cell sheet with large area, and provide enough mechanical support for the harvested cell sheet, and the cell sheet is used as a biological patch material for tissue repair, so that a bionic microenvironment can be provided for perfect regeneration of the injury, and a new idea can be provided for perfect repair of skin wound repair, hemostasis and cartilage defect.
Disclosure of Invention
The invention aims to provide a rapid harvesting method of a cell sheet layer and a 'colloid + cell sheet' composite film, the cell sheet layer and hydrogel are compounded and subjected to in-situ gelling, so that the rapid harvesting of a large-area cell sheet layer can be realized, and the obtained composite film compounded with the cell sheet layer and the hydrogel has good bioactivity, high mechanical strength, good tissue adhesion and good bionic effect, can be applied to the field of efficient tissue defect repair and regeneration, is perfectly integrated with surrounding tissues, and promotes tissue regeneration.
The technical scheme adopted by the invention is as follows:
a method of rapid harvesting of a cell sheet comprising: inoculating cells on the surface of a substrate, culturing to obtain a cell sheet layer, dripping a glue solution on the surface of the substrate, irradiating to ensure that the glue solution is subjected to photo-crosslinking, dripping excessive metal ion solution for ion crosslinking, and removing the excessive solution to easily remove the film layer compounded with the cell sheet layer to obtain the 'colloid + cell sheet' composite film.
In the above technical scheme, the glue solution is a mixed solution of sodium alginate Alg-NB modified by methyl N- (2-aminoethyl) -4- (4- (hydroxymethyl) -2-methoxy-5-nitrophenoxy) butyrate, gelatin GelMA grafted with methacrylic anhydride, and lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate LAP, which are stirred in the dark to be completely dissolved, and the metal ion is calcium ion.
Further, the mass ratio of GelMA, alg-NB and LAP is as follows: 5-20 wt%, 1-10 wt% and 0.05-0.4 wt%.
Furthermore, the mass ratio of the calcium ions to the GelMA is 10-30%.
Further, in the method: the glue solution is subjected to in-situ gelling through two steps of photo-crosslinking and ionic crosslinking, wherein the compounding effect of a colloid and a cell sheet layer can be improved by the characteristic of photo-aldehyde groups of light-responsive small molecules NB in the hydrogel, and the adhesion effect of the cell sheet layer and the surface of a substrate is weakened through ionic crosslinking, so that the easy desorption of the colloid and cell sheet composite film and the substrate is realized, and the glue solution can be used for rapidly harvesting large-area cell sheet layers.
Further, the cell is one or more of fibroblast, stem cell, osteoblast, osteoclast and chondrocyte.
Further, the substrate is one or more of silicon, quartz, titanium and silicon carbide with a surface subjected to pre-polishing treatment.
The prepared 'colloid + cell sheet' composite film can be used as a biological patch for tissue repair or regeneration, wherein the tissue is skin, cartilage or bone tissue.
Compared with the prior art, the invention has the following beneficial effects:
(1) According to the method, gelMA/Alg-NB/LAP hydrogel with good mechanical properties is innovatively utilized to harvest the cell sheet layer, the in-situ gel formation is realized through two steps of photo-crosslinking and ion crosslinking after the hydrogel is compounded with the cell sheet layer, the hydrogel and the cell sheet layer are rapidly shaped (the gel formation can be realized after about 1-3 s), the compounding effect of the gel and the cell sheet layer can be improved through the characteristic of photo-generated aldehyde groups of the photoresponse small molecule NB in the hydrogel, and more importantly, the ion crosslinking is innovatively introduced and applied to the desorption of the cell sheet layer, the binding force between the hydrogel/cell sheet layer and a culture substrate is greatly reduced in the ion crosslinking process, the mechanical properties of the hydrogel can also be greatly improved, and the easy harvesting of the cell sheet layer with large area can be realized.
(2) According to the invention, the hydrogel has good mechanical properties, tensile and pressure resistance, after the cell sheet layer is compounded, sufficient mechanical support can be provided for the cell sheet layer, the harvesting of a large-area cell sheet layer can be realized, the activity of cells and ECM active ingredients in the cell sheet layer can be well reserved for the harvested cell sheet layer, and on the other hand, the biological activity and biological functionality of the composite film formed by compounding the cell sheet layer into hydrogel are greatly improved due to the addition of the cell sheet layer. The obtained 'colloid + cell sheet' film is expected to be used as a biological patch and applied to skin wound repair, hemostasis and complete repair and regeneration of cartilage defect.
(3) The cell sheet layer used in the invention is obtained by in vitro culture, can realize large-scale preparation, is a cell membrane containing a large amount of ECM protein, and has good biological activity after being compounded with hydrogel, and the characteristic of the layered cell sheet has very important effects in specific application occasions, such as skin defect repair, osteochondral junction regeneration and the like.
Drawings
FIG. 1 is a schematic diagram of a preparation process of the present invention;
FIG. 2 shows the results of the rheological mechanical tests of the GelMA (5 wt%)/Alg-NB (1 wt%)/LAP (0.1 wt%) hydrogel of example 1;
FIG. 3 is a graph comparing the effects of GelMA/Alg-NB/LAP hydrogel desorbed from a PS substrate under photocrosslinking alone and under photocrosslinking + ionomeric crosslinking;
FIG. 4 is a photograph of hydrogel composite and harvested cell sheet of example 1, and the adhesion of the obtained "gel + cell sheet" to skin tissue;
Detailed Description
For better understanding of the present invention, the following further description is provided with reference to the accompanying drawings, but the embodiments of the present invention are not limited thereto.
GelMA, alg, NB and LAP are all already commercialized drugs, purchased directly from a specific company, and all drugs follow the principle of dry keeping in the dark.
The Alg-NB related in the invention can be obtained by preparation, and the specific method can be as follows:
dissolving 1g of Alg in 100mL of 0.01 mol/L2- (N-morphine) ethanesulfonic acid-hydrate (MES) buffer solution with pH =5.3, and reacting at 35 ℃; after the Alg is completely dissolved, 60mg of NB is dissolved in 10mL of dimethyl sulfoxide (DMSO), and the solution is added into the reaction system; adding 1.2g of 4- (4, 6-dimethoxytriazine-2-yl) -4-methylmorpholine hydrochloride (DMTMM) into the system in three times, wherein the interval is 0.5h each time, and finishing the reaction three hours after adding DMTMM for the last time; dialyzed against 0.1M NaCl solution (pH = 3.5) for 2d, then against deionized water for 2d, and freeze-dried to obtain Alg-NB. The invention provides a brand-new cell sheet harvesting method, which comprises the steps of injecting a specific colloidal solution onto a cultured cell sheet after in vitro culture to obtain a cell sheet with high cell density and rich in ECM, and adopting a two-step in-situ gel forming method of photocrosslinking and ionic crosslinking, so that the bonding force between the obtained composite film and a culture substrate is greatly reduced, and the composite film can be easily detached.
Example 1
1) GelMA (5 wt%), alg-NB (5 wt%) and LAP (0.1 wt%) are mixed according to a certain proportion, and stirred in the dark until the GelMA/Alg-NB/LAP glue is completely dissolved, so that GelMA/Alg-NB/LAP glue is obtained.
2) First, cells were cultured at 1X 10 5 cells/cm 2 Seeded on the surface of Si-substrate and cultured for 3 days to obtain a complete cell sheet.
3) After obtaining a complete cell sheet, the above-mentioned glue solution was dropped on the surface of the Si substrate, and then UV360-395 (50 mW/cm) 2 ) Completely gelatinizing after 0.1-10 min of illumination, and dripping excessive CaCl 2 Solution (CaCl) 2 Mass ratio to GelMA 20%) ion crosslinking was completed. Absorbing CaCl 2 And (5) slightly removing the hydrogel film compounded with the cell sheet layer from the solution. Thereby obtaining the 'colloid + cell sheet' composite filmAnd (3) a membrane.
The results of the rheological mechanical tests on the GelMA/Alg-NB/LAP hydrogel prepared in the example show that the gel forming time of the GelMA/Alg-NB/LAP hydrogel is 3.14s, the final storage modulus is 4542.3Pa (figure 2), and the results show that the hydrogel maintains the characteristic of quick gel forming and the mechanical properties are obviously improved. Compared with the effect that GelMA/Alg-NB/LAP hydrogel is desorbed from a PS substrate under the action of photocrosslinking and ionic crosslinking alone, the hydrogel residue on the PS substrate is much in only one-step photocrosslinking group, and the hydrogel residue on the PS substrate is almost not in the photocrosslinking and ionic crosslinking double crosslinking action, and the shape of the desorbed hydrogel is kept more complete (as shown in figure 3), which shows that the addition of ionic crosslinking is beneficial to reducing the adhesive force between the hydrogel and the substrate; in addition, as shown in fig. 4, the hydrogel can form a thin film on the Si substrate after photo-crosslinking and ion-crosslinking, after cell sheet layer recombination, the obtained "colloid + cell sheet" composite thin film can be easily detached from the substrate, and the detached "colloid + cell sheet" composite thin film has a certain tissue adhesion.
Example 2
1) A, mixing GelMA (20 wt%), alg-NB (10 wt%) and LAP (0.4 wt%) in proportion, and stirring in the dark until the GelMA/Alg-NB/LAP glue is completely dissolved to obtain the GelMA/Alg-NB/LAP glue.
2) First, cells were cultured at 1X 10 6 cells/cm 2 Seeded on the surface of Si-substrate and cultured for 3 days to obtain a complete cell sheet.
3) After obtaining a complete cell sheet, the above glue solution was dropped on the surface of the Si substrate, and then UV360-395 (100 mW/cm) 2 ) Completely gelatinizing after illumination for 10min, and dripping excessive CaCl 2 Solution (CaCl) 2 Mass ratio to GelMA 30%) ion crosslinking was completed. Absorbing CaCl 2 The solution is used for gently removing the 'molecular glue' compounded with the cell sheet layer. Thereby obtaining the 'colloid + cell sheet' composite film.
The result of the rheological mechanical test of the GelMA/Alg-NB/LAP hydrogel prepared in the embodiment shows that the gel forming time of the GelMA/Alg-NB/LAP is 3.45s, and the final storage modulus is 48965.3Pa, which indicates that the hydrogel maintains the characteristic of quick gel forming and the mechanical property of the hydrogel is obviously improved. Compared with the effect that GelMA/Alg-NB/LAP hydrogel is desorbed from a PS substrate under the action of photocrosslinking and ionic crosslinking alone, the hydrogel on the PS substrate is much remained only in the photocrosslinking group in one step, and under the action of photocrosslinking and ionic crosslinking, the hydrogel on the PS substrate is almost not remained, and the shape of the desorbed hydrogel is kept more complete, which shows that the addition of ionic crosslinking is beneficial to reducing the adhesive force between the hydrogel and the substrate; in addition, the hydrogel can form a film on a Si substrate after photo-crosslinking and ion crosslinking, the obtained 'colloid + cell sheet' composite film can be easily desorbed from the substrate after cell sheet layer compounding, and the desorbed 'colloid + cell sheet' composite film has certain tissue adhesion.
Example 3
1) GelMA (15 wt%), alg-NB (3 wt%) and LAP (0.05 wt%) are mixed according to a proportion, and stirred in the dark until the GelMA/Alg-NB/LAP glue is completely dissolved to obtain the GelMA/Alg-NB/LAP glue.
2) First, cells were cultured at 1X 10 3 cells/cm 2 Seeded on the substrate surface and cultured for 3 days to obtain a complete cell sheet.
3) After obtaining a complete cell sheet, the above-mentioned glue solution was dropped on the substrate surface, and then UV360-395 (1 mW/cm) 2 ) Completely gelatinizing after 0.1min of illumination, and dripping excessive CaCl 2 Solution (CaCl) 2 Mass ratio to GelMA of 10%) completed ionic crosslinking. Absorbing CaCl 2 The solution is used for gently removing the 'molecular glue' compounded with the cell sheet layer. Thereby obtaining the 'colloid + cell sheet' composite film.
The result of the rheological mechanical test of the GelMA/Alg-NB/LAP hydrogel prepared in the example shows that the gel forming time of the GelMA/Alg-NB/LAP is 2.98s, the final storage modulus is 25889.4Pa, and the result shows that the hydrogel maintains the characteristic of quick gel forming and the mechanical property of the hydrogel is obviously improved. Compared with the effect that GelMA/Alg-NB/LAP hydrogel is desorbed from a PS substrate under the action of photocrosslinking and ionic crosslinking alone, the hydrogel residue on the PS substrate is much in only one step of photocrosslinking group, and the hydrogel residue on the PS substrate is almost not in the action of photocrosslinking and ionic crosslinking, and the shape of the desorbed hydrogel is kept more complete, which shows that the addition of ionic crosslinking is beneficial to reducing the adhesive force between the hydrogel and the substrate; in addition, the hydrogel can form a film on a Si substrate after photo-crosslinking and ion crosslinking, the obtained 'colloid + cell sheet' composite film can be easily desorbed from the substrate after cell sheet layer compounding, and the desorbed 'colloid + cell sheet' composite film has certain tissue adhesion.
Claims (9)
1. A method for rapid harvesting of a cell sheet, comprising: inoculating cells on the surface of a substrate, culturing to obtain a cell sheet layer, dripping a glue solution on the surface of the substrate, irradiating to ensure that the glue solution is subjected to photo-crosslinking, dripping excessive metal ion solution for ion crosslinking, and removing the excessive solution to easily remove the film layer compounded with the cell sheet layer to obtain the 'colloid + cell sheet' composite film.
2. The method for rapidly harvesting the cell sheet layer as claimed in claim 1, wherein the glue solution is a mixed solution of sodium alginate Alg-NB modified by methyl N- (2-aminoethyl) -4- (4- (hydroxymethyl) -2-methoxy-5-nitrophenoxy) butyrate, gelatin GelMA grafted by methacrylic anhydride, and lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate LAP which is stirred in the dark to be completely dissolved, and the metal ion is calcium ion.
3. The method for rapidly harvesting the cell sheet according to claim 2, wherein the mass ratio of GelMA, alg-NB and LAP is as follows: 5-20 wt%, 1-10 wt% and 0.05-0.4 wt%.
4. The method for rapidly harvesting a cell sheet according to claim 2, wherein the mass ratio of the calcium ions to the GelMA is 10-30%.
5. The method for rapidly harvesting the cell sheet according to claim 1, wherein the glue solution is subjected to in-situ gel formation through two steps of photo-crosslinking and ion crosslinking, wherein the compounding effect of colloids and the cell sheet can be improved through the characteristic of photo-generated aldehyde groups of light-responsive small molecules NB in the hydrogel, and the adhesion between the cell sheet and the surface of the substrate is weakened through the ion crosslinking, so that the colloid + cell sheet composite film and the substrate can be easily desorbed, and the method can be used for rapidly harvesting large-area cell sheets.
6. The method for rapidly harvesting a cell sheet according to claim 1, wherein the cells are one or more of fibroblasts, stem cells, osteoblasts, osteoclasts, and chondrocytes.
7. The rapid harvesting method of the cell sheet according to claim 1, wherein the substrate is one or more of silicon, quartz, titanium, and silicon carbide with a surface that is pre-polished.
8. A "colloid + cell sheet" composite film, which is prepared by the method of any one of claims 1 to 7.
9. Use of the composite film of claim 8 as a biological patch in tissue repair or regeneration, wherein the tissue is skin, cartilage, or bone.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007050314A2 (en) * | 2005-10-24 | 2007-05-03 | Hosheng Tu | Medical device with living cell sheet |
| CN105169487A (en) * | 2015-10-27 | 2015-12-23 | 上海交通大学 | Wound healing cell sheet with biological activity, preparing method and application |
| CN106924817A (en) * | 2017-03-02 | 2017-07-07 | 浙江大学 | A kind of ultra-thin carrier cell piece and preparation method thereof |
| CN106963987A (en) * | 2017-03-29 | 2017-07-21 | 浙江大学 | A kind of conductive extracellular matrix laminated film obtained via cell sheets and preparation method thereof |
| CN112292447A (en) * | 2019-02-28 | 2021-01-29 | 京东方科技集团股份有限公司 | Umbilical cord mesenchymal stem cell and preparation method of cell membrane thereof |
| CN112898599A (en) * | 2021-02-02 | 2021-06-04 | 深圳市第二人民医院(深圳市转化医学研究院) | Three-dimensional network bionic hydrogel and preparation method and application thereof |
-
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007050314A2 (en) * | 2005-10-24 | 2007-05-03 | Hosheng Tu | Medical device with living cell sheet |
| CN105169487A (en) * | 2015-10-27 | 2015-12-23 | 上海交通大学 | Wound healing cell sheet with biological activity, preparing method and application |
| CN106924817A (en) * | 2017-03-02 | 2017-07-07 | 浙江大学 | A kind of ultra-thin carrier cell piece and preparation method thereof |
| CN106963987A (en) * | 2017-03-29 | 2017-07-21 | 浙江大学 | A kind of conductive extracellular matrix laminated film obtained via cell sheets and preparation method thereof |
| CN112292447A (en) * | 2019-02-28 | 2021-01-29 | 京东方科技集团股份有限公司 | Umbilical cord mesenchymal stem cell and preparation method of cell membrane thereof |
| CN112898599A (en) * | 2021-02-02 | 2021-06-04 | 深圳市第二人民医院(深圳市转化医学研究院) | Three-dimensional network bionic hydrogel and preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| XIAOJUN LONG, 等: "Biomimetic macroporous hydrogel with a triple-network structure for full-thickness skin regeneration", APPLIED MATERIALS TODAY, vol. 27, pages 101442 * |
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