CN115645628B - Harvesting method of cell sheet layer and composite film of colloid and cell sheet - Google Patents
Harvesting method of cell sheet layer and composite film of colloid and cell sheet Download PDFInfo
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- 238000003306 harvesting Methods 0.000 title claims abstract description 22
- 238000000034 method Methods 0.000 title claims abstract description 22
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- 238000004132 cross linking Methods 0.000 claims abstract description 54
- 239000000017 hydrogel Substances 0.000 claims abstract description 50
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- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical class CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 2
- 125000000022 2-aminoethyl group Chemical group [H]C([*])([H])C([H])([H])N([H])[H] 0.000 claims description 2
- 108010010803 Gelatin Proteins 0.000 claims description 2
- RTAQQCXQSZGOHL-UHFFFAOYSA-N Titanium Chemical compound [Ti] RTAQQCXQSZGOHL-UHFFFAOYSA-N 0.000 claims description 2
- ANCHPZQGZBCDBK-UHFFFAOYSA-N [PH2](O)=O.C1(=CC=CC=C1)C=1C(=C(C(=O)[Li])C(=CC1C)C)C Chemical compound [PH2](O)=O.C1(=CC=CC=C1)C=1C(=C(C(=O)[Li])C(=CC1C)C)C ANCHPZQGZBCDBK-UHFFFAOYSA-N 0.000 claims description 2
- 230000015572 biosynthetic process Effects 0.000 claims description 2
- PWLNAUNEAKQYLH-UHFFFAOYSA-N butyric acid octyl ester Natural products CCCCCCCCOC(=O)CCC PWLNAUNEAKQYLH-UHFFFAOYSA-N 0.000 claims description 2
- 210000001612 chondrocyte Anatomy 0.000 claims description 2
- 238000003795 desorption Methods 0.000 claims description 2
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- 235000019322 gelatine Nutrition 0.000 claims description 2
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- DCUFMVPCXCSVNP-UHFFFAOYSA-N methacrylic anhydride Chemical compound CC(=C)C(=O)OC(=O)C(C)=C DCUFMVPCXCSVNP-UHFFFAOYSA-N 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims description 2
- UUIQMZJEGPQKFD-UHFFFAOYSA-N n-butyric acid methyl ester Natural products CCCC(=O)OC UUIQMZJEGPQKFD-UHFFFAOYSA-N 0.000 claims description 2
- 210000000963 osteoblast Anatomy 0.000 claims description 2
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- 239000010703 silicon Substances 0.000 claims description 2
- HBMJWWWQQXIZIP-UHFFFAOYSA-N silicon carbide Chemical compound [Si+]#[C-] HBMJWWWQQXIZIP-UHFFFAOYSA-N 0.000 claims description 2
- 229910010271 silicon carbide Inorganic materials 0.000 claims description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 2
- 239000010936 titanium Substances 0.000 claims description 2
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- 230000002508 compound effect Effects 0.000 claims 1
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- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
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- ZYNNVAZLFGFFLF-UHFFFAOYSA-N ethanesulfonic acid hydrate Chemical compound O.CCS(O)(=O)=O.CCS(O)(=O)=O ZYNNVAZLFGFFLF-UHFFFAOYSA-N 0.000 description 2
- 239000011664 nicotinic acid Substances 0.000 description 2
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- MIJPAVRNWPDMOR-ZAFYKAAXSA-N L-ascorbic acid 2-phosphate Chemical compound OC[C@H](O)[C@H]1OC(=O)C(OP(O)(O)=O)=C1O MIJPAVRNWPDMOR-ZAFYKAAXSA-N 0.000 description 1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- Materials For Medical Uses (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a rapid harvesting method of a cell sheet layer and a 'colloid and cell sheet' composite film, which comprises the steps of inoculating cells to the surface of a substrate, culturing to obtain the cell sheet layer, dripping a glue solution on the surface of the substrate, photo-crosslinking the glue solution by illumination, dripping excessive metal ion solution for ion crosslinking, and removing the redundant solution by sucking, so that the film layer compounded with the cell sheet layer can be easily removed to obtain the 'colloid and cell sheet' composite film. According to the method, the glue material and the cell sheet layer can be fully compounded, photo-crosslinking and ion crosslinking can obtain hydrogel with good mechanical properties, the property of photo-generated aldehyde groups of the hydrogel can improve the compounding level of colloid and the cell sheet layer, the binding force between a colloid and cell sheet film and a substrate in the ion crosslinking process is greatly reduced, the harvesting of the cell sheet layer with a large area and the compounding of the colloid and cell sheet film with the hydrogel can be realized, and the obtained colloid and cell sheet film is expected to be used as a biological patch and applied to the repair of skin wounds, 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 biomedical field, in particular to a method for harvesting cell sheets and compounding hydrogel, which can realize the harvesting of large-area cell sheets and the addition of the cell sheets can promote the bioactivity of hydrogel scaffold materials.
Background
In recent years, cell sheet technology has been widely used in the field of tissue engineering [ Long T. The Effect of Mesenchymal Stem Cell Sheets on Structural Allograft Healing of Critical Sized Femoral Defects in Mice. Biomaterials 2014;35:2752-2759] because it retains the advantages of high density of cellular tissue, natural ECM secreted by mature cells, and being able to be engineered. For example, cell sheet technology is believed to offer a potential solution for skin repair to reduce scarring even to a perfectly regenerative state. Cheng et al rapidly prepared adipose-derived stem cell (ASC) sheet by stimulation with L-ascorbic acid 2-phosphate and applied it to the fields of tissue regeneration and wound healing, and studies showed that ASC sheet has the necessary secretion factors, can improve skin wound healing, and can generate new skin with excellent quality [ Yu J. Cell Sheet Composed of Adipose-Derived Stem Cells Demonstrates Enhanced Skin Wound Healing with Reduced Scar formation, acta biomaterials 2018;77:191-200]. The cell sheet harvesting technology is also very important, but the cell sheet obtained in the prior art has the defects of poor mechanical strength, difficult acquisition of a large-area cell sheet and the like, which restricts the application prospect in the repair of full-layer skin injury.
Based on the above, taking into consideration the characteristics of good mechanical properties of the hydrogel material, namely good biocompatibility and biodegradability [ Ghobril C. The Chemistry and Engineering of Polymeric Hydrogel Adhesives for Wound Closure: a Tutorial. Chemical Society Reviews 2015;44:1820-1835], if the cell sheet layer can be harvested by using the hydrogel with good mechanical strength and the cell sheet layer with high biological activity is compounded with the hydrogel, it is possible to harvest the cell sheet layer with large area, and provide sufficient mechanical support for the harvested cell sheet layer, and the harvested cell sheet layer can be used as a biological patch material for tissue repair, can provide a bionic microenvironment for perfect regeneration of a damaged part, and can provide a new idea for skin wound repair, hemostasis and perfect repair of cartilage defects.
Disclosure of Invention
The invention aims to provide a rapid harvesting method of a cell sheet layer and a colloid and cell sheet composite film, wherein the rapid harvesting of the cell sheet layer in a large area can be realized by compositing the cell sheet layer with hydrogel and forming the gel in situ, and the obtained composite film composited 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 fields of repairing and regenerating high-efficiency tissue defects, 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 cell sheets, comprising: inoculating cells to the surface of a substrate, culturing to obtain a cell sheet layer, dripping a glue solution on the surface of the substrate, photo-crosslinking the glue solution by illumination, dripping excessive metal ion solution for ion crosslinking, and removing the excessive solution by suction to easily tear off the film layer compounded with the cell sheet layer to obtain the colloid and cell sheet composite film.
In the above technical scheme, the glue solution is a mixed solution of N- (2-aminoethyl) -4- (4- (hydroxymethyl) -2-methoxy-5-nitrophenoxy) methyl butyrate modified sodium alginate Alg-NB, methacrylic anhydride grafted gelatin GelMA and phenyl-2, 4, 6-trimethylbenzoyl lithium phosphinate LAP which are stirred in the dark until 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% of the material, 1-10 wt% of the material, and 0.05-0.4 wt% of the material.
Further, the mass ratio of the calcium ions to the GelMA is 10% -30%.
Further, in the method: the glue solution is subjected to two-step in-situ glue formation through photocrosslinking and ionic crosslinking, wherein the characteristic of photo-induced aldehyde groups of the photoresponsive micromolecule NB in the hydrogel can improve the composite effect of colloid and cell sheet layers, and the ionic crosslinking weakens the adhesion effect of the cell sheet layers and the surface of the substrate, so that the 'colloid+cell sheet' composite film and the substrate are easily desorbed, and the glue solution can be used for rapidly harvesting large-area cell sheet layers.
Further, the cells are one or more of fibroblasts, stem cells, osteoblasts, osteoclasts and chondrocytes.
Further, the substrate is one or more of silicon, quartz, titanium and silicon carbide with the surface being subjected to pre-polishing treatment.
The prepared gel and 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) The GelMA/Alg-NB/LAP hydrogel with good mechanical properties is innovatively utilized to harvest the cell sheets, after the cell sheets are composited by the hydrogel, in-situ gel forming is realized by two steps of photocrosslinking and ion crosslinking, so that the hydrogel and the cell sheets are rapidly shaped (about 1-3s can be crosslinked), the characteristic of photocrosslinking small molecular NB in the hydrogel can promote the compositing effect of the gel and the cell sheets, and more importantly, the novel ionic crosslinking is introduced to be applied to desorption of the cell sheets, the binding force between the hydrogel/cell sheets and a culture substrate is greatly reduced in the ionic crosslinking process, the mechanical properties of the hydrogel are also greatly improved, and the easy harvesting of the cell sheets with large area can be realized.
(2) In the invention, the hydrogel has good mechanical properties, tensile and pressure resistance, after the cell sheet is compounded, enough mechanical support can be provided for the cell sheet, the harvesting of the cell sheet with a large area can be realized, the harvested cell sheet can well retain the activity of cells and ECM active components in the cell sheet, and on the other hand, the biological activity and the biological functionality of the composite film formed by compounding the cell sheet with the water-feeding gel are greatly improved due to the addition of the cell sheet. The obtained gel and 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 defects.
(3) In the invention, the cell sheet layer is obtained through in vitro culture, so that large-scale preparation can be realized, the cell sheet layer is a cell film containing a large amount of ECM proteins, the hydrogel/cell sheet layer obtained after the hydrogel is compounded has good bioactivity, and the characteristic of lamellar cell sheets has very important effects in specific application occasions, such as skin defect repair, osteochondral connection regeneration and the like.
Drawings
FIG. 1 is a schematic illustration of a preparation flow of the present invention;
FIG. 2 is the results of a rheological mechanical test 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 hydrogels detached from PS substrates under photocrosslinking alone and photocrosslinking+ion crosslinking in combination;
FIG. 4 is a photograph showing the hydrogel composition and harvesting of cell sheets of example 1, and the resulting adhesion of "gel+cell sheets" to skin tissue;
description of the embodiments
For a better understanding of the present invention, the following description of the present invention will be given with reference to the accompanying drawings, but the embodiments of the present invention are not limited thereto.
GelMA, alg, NB and LAP are all commercial medicines, and are purchased directly from specific companies, and all medicines follow the principle of keeping the medicines dry in the dark.
The Alg-NB related in the invention can be obtained by preparation, and the specific method comprises the following steps:
1 g of Alg was dissolved in 100 mL of 0.01 mol/L2- (N-morphine) ethanesulfonic acid-hydrate (MES) buffer solution at pH=5.3 and reacted at 35 ℃; dissolving 60 mg of NB in 10mL of dimethyl sulfoxide (DMSO) after Alg is completely dissolved, and adding into a reaction system; 1.2 g of 4- (4, 6-dimethoxy triazine-2-yl) -4-methylmorpholine hydrochloride (DMTMM) is added into the system in three times, each time interval is 0.5 h, and the reaction is ended three hours after the DMTMM is added for the last time; dialysis was performed with 0.1M NaCl solution (ph=3.5) for 2 d, then with deionized water for 2 d, and freeze-drying to obtain Alg-NB. The invention provides a brand new method for harvesting cell sheets, which comprises the steps of injecting a specific colloidal solution into the cultured cell sheets after obtaining the cell sheets with high cell density and rich ECM (electro-magnetic memory), and adopting a two-step in-situ gel forming method sequentially carried out by photocrosslinking and ion crosslinking, so that the binding force between the obtained composite film and a culture substrate is greatly reduced, and the method can be used for easily desorbing.
Examples
1) GelMA (5 wt%), alg-NB (5 wt%) and LAP (0.1 wt%) were mixed in proportion and stirred in the dark until completely dissolved to obtain GelMA/Alg-NB/LAP glue.
2) First, the cells were mixed at 1X 10 5 cells/cm 2 Is inoculated on the surface of the 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 onto the Si substrate surface, and then UV360-395 (50 mW/cm 2 ) After illumination for 0.1-10 min, completely gelling, and dripping excess CaCl 2 Solution (CaCl) 2 20% by mass with GelMA). Sucking CaCl 2 The solution gently peels off the hydrogel film compounded with the cell sheet layer. Thus obtaining the composite film of the colloid and the cell slice.
The rheological mechanical test result of the GelMA/Alg-NB/LAP hydrogel prepared in the example shows that the gel forming time of the GelMA/Alg-NB/LAP is 3.14s, the final storage modulus is 4542.3 Pa (figure 2), and the result shows that the hydrogel maintains the characteristic of rapid gel forming and the mechanical property of the gel is remarkably improved. Comparing the effect of GelMA/Alg-NB/LAP hydrogel detached from PS substrate under photo-crosslinking effect and photo-crosslinking and ion crosslinking double-crosslinking effect alone, it can be seen that there is only one photo-crosslinking group, hydrogel residue on PS substrate is much, and hydrogel residue on PS substrate is almost not left under photo-crosslinking and ion crosslinking double-crosslinking effect, and the shape of the desorbed hydrogel is kept more complete (as shown in figure 3), which shows that the addition of ion crosslinking helps to reduce the adhesion of hydrogel and substrate; in addition, as shown in fig. 4, the hydrogel can form a film on the Si substrate after photo-crosslinking and ion-crosslinking, and after the cell sheet layer is compounded, the obtained "colloid+cell sheet" composite film can be easily detached from the substrate, and the detached "colloid+cell sheet" composite film has a certain tissue adhesion.
Examples
1) A, gelMA (20 wt%), alg-NB (10 wt%) and LAP (0.4 wt%) are mixed according to a proportion, and stirred in the dark until the GelMA/Alg-NB/LAP glue is completely dissolved.
2) First, the cells were mixed at 1X 10 6 cells/cm 2 Is inoculated on the surface of the 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 onto the Si substrate surface, and then UV360-395 (100 mW/cm 2 ) Completely gelling after illumination for 10min, and dripping excessive CaCl 2 Solution (CaCl) 2 30% by mass with GelMA) to complete the ionic crosslinking. Sucking CaCl 2 The solution gently strips the "molecular glue" that has compounded the cell sheet. Thus obtaining the composite film of the colloid and the cell slice.
The rheological mechanical test result of the GelMA/Alg-NB/LAP hydrogel prepared in the example shows that the gel forming time of the GelMA/Alg-NB/LAP is 3.45s, and the final storage modulus is 48965.3Pa, so that the result shows that the hydrogel maintains the characteristic of rapid gel forming, and the mechanical property of the gel is remarkably improved. Compared with the effect that GelMA/Alg-NB/LAP hydrogel is singly desorbed from the PS substrate under the photo-crosslinking effect and the photo-crosslinking and ion-crosslinking double-crosslinking effect, only one photo-crosslinking group exists, the hydrogel residue on the PS substrate is much, the hydrogel residue on the PS substrate is almost not left under the photo-crosslinking and ion-crosslinking double-crosslinking effect, the shape of the desorbed hydrogel is kept more complete, and the addition of the ion crosslinking is helpful for reducing the adhesion of the hydrogel and the substrate; in addition, the hydrogel can form a film on the Si substrate after photo-crosslinking and ion crosslinking, and after the cell sheet layer is compounded, the obtained 'colloid and cell sheet' compound film can be easily detached from the substrate, and the detached 'colloid and cell sheet' compound film has certain tissue adhesion.
Examples
1) GelMA (15 wt%), alg-NB (3 wt%) and LAP (0.05 wt%) were mixed in proportion and stirred in the dark until completely dissolved to obtain GelMA/Alg-NB/LAP glue.
2) First, the cells were mixed at 1X 10 3 cells/cm 2 Is inoculated on the substrate surface and cultured for 3 days to obtain a complete cell sheet.
3) After obtaining a complete cell sheet, the above glue solution was dropped onto the substrate surface, followed by UV360-395 (1 mW/cm 2 ) Completely gelling after illumination for 0.1min, and dripping excessive CaCl 2 Solution (CaCl) 2 The mass ratio of the polymer to GelMA is 10 percent) to finish ionic crosslinking. Sucking CaCl 2 The solution gently strips the "molecular glue" that has compounded the cell sheet. Thus obtaining the composite film of the colloid and the cell slice.
The rheological mechanical test result 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, and the final storage modulus is 25889.4Pa, so that the result shows that the hydrogel maintains the characteristic of rapid gel forming, and the mechanical property of the gel is remarkably improved. Compared with the effect that GelMA/Alg-NB/LAP hydrogel is singly desorbed from the PS substrate under the photo-crosslinking effect and the photo-crosslinking and ion-crosslinking double-crosslinking effect, only one photo-crosslinking group exists, the hydrogel residue on the PS substrate is much, the hydrogel residue on the PS substrate is almost not left under the photo-crosslinking and ion-crosslinking double-crosslinking effect, the shape of the desorbed hydrogel is kept more complete, and the addition of the ion crosslinking is helpful for reducing the adhesion of the hydrogel and the substrate; in addition, the hydrogel can form a film on the Si substrate after photo-crosslinking and ion crosslinking, and after the cell sheet layer is compounded, the obtained 'colloid and cell sheet' compound film can be easily detached from the substrate, and the detached 'colloid and cell sheet' compound film has certain tissue adhesion.
Claims (7)
1. A method of harvesting a cell sheet, comprising: inoculating cells to the surface of a substrate, culturing to obtain a cell sheet layer, dripping a glue solution on the surface of the substrate, photo-crosslinking the glue solution by illumination, dripping excessive metal ion solution for ion crosslinking, sucking the excessive solution, and removing the film layer compounded with the cell sheet layer to obtain a colloid and cell sheet composite film;
the glue solution is a mixed solution of N- (2-aminoethyl) -4- (4- (hydroxymethyl) -2-methoxy-5-nitrophenoxy) methyl butyrate modified sodium alginate Alg-NB, methacrylic anhydride grafted gelatin GelMA and phenyl-2, 4, 6-trimethylbenzoyl lithium phosphinate LAP which are stirred in the dark until the solution is completely dissolved, and the metal ions are calcium ions.
2. The method for harvesting cell sheets according to claim 1, wherein the mass ratio of GelMA, alg-NB, LAP is: 5-20 wt% of the material, 1-10 wt% of the material, and 0.05-0.4 wt% of the material.
3. The method for harvesting the cell sheets according to claim 1, wherein the mass ratio of calcium ions to GelMA is 0.1-0.3: 1.
4. the method for harvesting cell sheets according to claim 1, wherein the glue solution is subjected to two-step in-situ glue formation by photo-crosslinking and ion crosslinking, wherein the property of photo-induced aldehyde groups of the photo-responsive small molecules NB in the hydrogel can promote the compound effect of the colloid and the cell sheets, and the ion crosslinking weakens the adhesion effect of the cell sheets and the surface of the substrate, so that the desorption of the compound film of colloid and cell sheets from the substrate is realized, and the method can be used for harvesting large-area cell sheets.
5. The method of claim 1, wherein the cells are one or more of fibroblasts, stem cells, osteoblasts, osteoclasts, and chondrocytes.
6. The method according to claim 1, wherein the substrate is one of silicon, quartz, titanium, and silicon carbide with a surface polished in advance.
7. A "gel+cell sheet" composite film prepared by the method of any one of claims 1-6.
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