CN114292808B - Preparation method of polysaccharide scaffold material, and product and application thereof - Google Patents
Preparation method of polysaccharide scaffold material, and product and application thereof Download PDFInfo
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Abstract
The invention discloses a preparation method of a polysaccharide scaffold material, a product and application thereof, and relates to the technical field of in-vitro large-scale culture of cells. The preparation method comprises the following steps: (1) Freeze-drying plant tissues, and then soaking and washing the plant tissues with ultrapure water to obtain soaked and washed plant tissues; (2) And carrying out RGD sequence covalent bond derivatization and/or collagen adsorption fibrosis treatment on the plant tissues after infiltration and washing, and finally, carrying out freeze-drying to obtain the polysaccharide scaffold material. According to the invention, the plant polysaccharide material is subjected to comparative analysis, so that a stent material source suitable for development is optimized, the surface of the plant polysaccharide material is further modified by a stable chemical or physical treatment method, and the polysaccharide stent material is prepared, has good biocompatibility, and can be used for cell expansion in various cell culture devices such as a culture dish, a bottle, a biological reaction container and the like.
Description
Technical Field
The invention relates to the technical field of in-vitro large-scale culture of cells, in particular to a preparation method of a polysaccharide scaffold material, a product and application thereof.
Background
In recent years, with the rapid development of biotechnology, new technologies such as biomedical treatment and cell agriculture based on cell culture are rapidly raised, and the rapid development is presented, and the new technologies are listed as strategic front technologies by the national institutes of science and technology, the national institutes of america and the like, and become important fields for global high-tech research and development.
Wherein large scale cell culture is one of the key technologies for which a need exists. Current cell mass culture techniques are constructed with carriers, which are usually based on animal collagen with good biocompatibility, such as Cytodex commercial microcarriers produced by GE. In addition, there are commercial microcarriers produced using polyester materials as a basis, such as those produced by Sidoris.
Cellulose is a natural polysaccharide, and is considered as a potential scaffold material for cell culture due to the characteristics of good biocompatibility, rich sources and the like. The research is generally based on high-purity bacterial cellulose, and then the biocompatibility of the bacterial cellulose is improved through further physical or chemical modification, so that the bacterial cellulose is used in the fields of cell culture or regenerative medicine and the like. However, in cellular agriculture such as research on cultivated meat (bio-cultivated meat/cell cultivated meat), there is a higher level of demand for edible, low-cost and high-efficiency development of scaffold materials, and natural plant cellulose, which is abundant in source, is a lower-cost source of scaffold materials. In recent years, research and development of plant cellulose application, such as apples, leaves, celery and the like, are also developed in the field, and a development foundation of plant polysaccharide scaffold materials is laid.
However, natural plant-based cellulose has several distinct features compared to bacterial cellulose: ① Purity, natural plant-based cellulose contains lignin, polysaccharide and other impurity components; ② The structure, natural plant-based cellulose has a definite three-dimensional structure, and has stronger rigidity, such as hemp, cotton, wood and the like; ③ The biocompatibility is not high, and the natural plant-based cellulose has a large number of negative groups on the surface and contains impurities, so that the biocompatibility is not high; ④ The pretreatment is complex, and the pulping and purifying processes of the raw materials are complex. These factors have led to limited application in the life sciences.
Disclosure of Invention
The invention aims to provide a preparation method of polysaccharide scaffold material, a product and application thereof, so as to solve the problems in the prior art.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a preparation method of a polysaccharide scaffold material, which comprises the following steps:
(1) Freeze-drying plant tissues, and then soaking and washing the plant tissues with ultrapure water to obtain soaked and washed plant tissues;
(2) And carrying out RGD sequence covalent bond derivatization and/or collagen adsorption fibrosis treatment on the plant tissues after infiltration and washing, and finally, carrying out freeze-drying to obtain the polysaccharide scaffold material.
Further, in the step (1), the plant tissue is fresh vegetable and fruit tissue.
Further, in step (1), the conditions of the lyophilization process are: the freezing temperature is-20 to-30 ℃, the vacuum degree is 0.20 to 1.00mbar, and the drying time is 18 to 24 hours.
Further, in step (2), the lyophilization conditions are: the freezing temperature is-20 to-30 ℃, the vacuum degree is 0.20 to 1.00mbar, and the drying time is 24 to 36 hours.
Further, in step (2), the method for treating covalent bond derivatization of the RGD sequence comprises: the infiltrated and washed plant tissue is reacted with N-hydroxysuccinimide in the presence of EDC in dimethylformamide solution, and RGD sequence is added to dimethylformamide solution and further reacted.
Further, the RGD sequence is Ac-Arg-Gly-Asp-Ser-Lys-NH 2.
Further, in the step (2), the treatment method of collagen adsorption fibrosis comprises: immersing the plant tissues subjected to the immersion washing into a collagen solution for immersion incubation, then adjusting the collagen solution to be pH=7, and finally immersing and washing with PBS.
The invention also provides the polysaccharide stent material prepared by the preparation method.
The invention also provides application of the polysaccharide scaffold material in cell culture.
Further, the cells are chicken muscle stem cells or chicken fat precursor cells.
The invention discloses the following technical effects:
the polysaccharide scaffold material prepared by the invention has the following advantages:
① The polysaccharide scaffold material has a rigid three-dimensional hole structure, large surface area, controllable appearance size and structure and pore diameter of 25-150 mu m, and is convenient for nutrient substance transmission and metabolic substance diffusion and cell adhesion;
② The polysaccharide stent material has definite treatment process, can be prepared in a standardized way, is natural in source, is introduced with no toxic and harmful substances, and has edible natural properties;
③ The rigid structure provided by the polysaccharide scaffold material is favorable for directional arrangement of muscle stem cells, so that directional fusion of the muscle stem cells is facilitated to form myotube cells;
④ The polysaccharide scaffold material has good biocompatibility, can be used for large-scale expansion culture of animal cells such as chicken muscle stem cells and chicken fat precursor cells, and has a cell culture density of 1X 10 7/mL;
⑤ The polysaccharide scaffold material has rich raw material sources and low cost, and can be popularized and applied in the field of cell agriculture in a large scale and at low cost.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing growth of chicken muscle stem cells in polysaccharide scaffold material prepared in example 3;
FIG. 2 is a graph showing the growth of chicken fat precursor cells in the polysaccharide scaffold material prepared in example 3;
FIG. 3 is a schematic diagram showing growth curves of chicken muscle stem cells in polysaccharide scaffold materials;
FIG. 4 is a schematic diagram showing growth curves of chicken fat precursor cells in polysaccharide scaffold materials.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
In the following examples or comparative examples:
the preparation method of the SDS solution comprises the following steps: 5.00g of SDS (sodium dodecyl sulfate) is weighed and dissolved in 5000mL of deionized water, and the mixture is stirred uniformly by a glass rod to obtain 0.1% SDS solution;
The PBS preparation method comprises the following steps: 8g of NaCl, 0.2g of KCL and 0.24g of Na 2HPO4·12H2O 3.63g,KH2PO4 are weighed and dissolved in 900mL of double distilled water, the pH value is regulated to 7.4 by hydrochloric acid, and water is added to fix the volume to 1L. Sterilizing under high pressure (at least 20 min), and storing at room temperature or 4deg.C in refrigerator;
The preparation method of the collagen solution comprises the following steps: 500uL of bovine collagen (6.0 mg/mL, sigma) solution was pipetted into a test tube, then 500uL of 0.1mol/L acetic acid was pipetted again, and the mixture was gently blown and mixed to obtain 3.0mg/mL collagen solution.
Example 1
(1) Raw material selection: selecting fresh carrots;
(2) And (3) material treatment: cleaning radix Dauci Sativae, and peeling;
(3) And (3) material segmentation: cutting peeled radix Dauci Sativae into 1cm 3 square tissue blocks (or any other required specification) according to longitudinal direction or transverse direction;
(4) And (3) freeze drying: placing the tissue block in a freeze dryer at a freezing temperature of: -20 ℃, vacuum degree is: 1.00mbar, drying time 24h;
(5) Soaking and washing: immersing the freeze-dried tissue blocks in ultrapure water, shaking for 24 hours by using a shaking table, replacing ultrapure water, repeating for three times, and then placing the tissue blocks in a freeze dryer to obtain the polysaccharide scaffold material after freeze drying, wherein the freezing temperature is as follows: -20 ℃, vacuum degree is: 1.00mbar, drying time 36h;
(6) Collagen fibrosis adsorption treatment: immersing the freeze-dried polysaccharide scaffold material into a collagen solution of 3.0mg/mL, immersing and incubating for 50h at 4 ℃, adjusting the pH value of the collagen solution to be 7 by using a NaOH solution, immersing and cleaning for 12h by using PBS, and then placing the tissue block into a freeze dryer, and obtaining the collagen fibrosis adsorption polysaccharide scaffold material after freeze drying, wherein the freezing temperature is as follows: -20 ℃, vacuum degree is: 1.00mbar, drying time 36h;
(7) And (3) sterilization: and carrying out double-layer sealing packaging and irradiation sterilization on the prepared polysaccharide stent material.
Example 2
(1) Raw material selection: selecting fresh carrots;
(2) And (3) material treatment: cleaning radix Dauci Sativae, and peeling;
(3) And (3) material segmentation: cutting peeled radix Dauci Sativae into 1cm 3 square tissue blocks (or any other required specification) according to longitudinal direction or transverse direction;
(4) And (3) freeze drying: placing the tissue block in a freeze dryer at a freezing temperature of: -30 ℃, vacuum degree is: 0.20mbar, drying time 18h;
(5) Soaking and washing: immersing the freeze-dried tissue blocks in ultrapure water, shaking the tissue blocks for 24 hours by using a shaking table, replacing the ultrapure water, and repeating the steps for three times;
(6) Covalent bond derivatization treatment of RGD sequences:
The washed tissue mass (101 mg,660 nmol) was infiltrated with N-hydroxysuccinimide (230 mg,2000 nmol) in 5mL of Dimethylformamide (DMF) in the presence of EDC (190 mg,991 nmol) for 2 hours at 0℃and then Ac-Arg-Gly-Asp-Ser-Lys-NH 2 (400 mg,660 nmol) was added and further reacted with shaking for 16 hours, followed by washing with pure water three times each for 12 hours, and then the tissue mass was placed in a freeze-dryer and freeze-dried to obtain RGD sequence covalent bond-derived polysaccharide scaffold material, wherein the freezing temperature was: -20 ℃, vacuum degree is: 1.00mbar, drying time 36h;
(7) And (3) sterilization: and carrying out double-layer sealing packaging and irradiation sterilization on the prepared polysaccharide stent material.
Example 3
(1) Raw material selection: selecting fresh carrots;
(2) And (3) material treatment: cleaning radix Dauci Sativae, and peeling;
(3) And (3) material segmentation: cutting peeled radix Dauci Sativae into 1cm 3 square tissue blocks or other arbitrary specifications according to longitudinal direction or transverse direction;
(4) And (3) freeze drying: placing the tissue block in a freeze dryer at a freezing temperature of: -20 ℃, vacuum degree is: 1.00mbar, drying time 24h;
(5) Soaking and washing: immersing the freeze-dried tissue blocks in ultrapure water, shaking the tissue blocks for 24 hours by using a shaking table, replacing the ultrapure water, and repeating the steps for three times;
(6) Covalent bond derivatization treatment of RGD sequences:
the washed tissue pieces (101 mg,660 nmol) were infiltrated and reacted with N-hydroxysuccinimide (230 mg,2000 nmol) in 5mL of Dimethylformamide (DMF) at 0℃for 2h in the presence of EDC (190 mg,991 nmol). Then adding Ac-Arg-Gly-Asp-Ser-Lys-NH 2 (400 mg,660 nmol) and further vibrating for reaction for 16 hours, soaking and cleaning with pure water for three times each time for 12 hours, then placing the tissue block in a freeze dryer, and obtaining the RGD sequence covalent bond derivative polysaccharide scaffold material after freeze drying, wherein the freezing temperature is as follows: -20 ℃, vacuum degree is: 1.00mbar, drying time 36h;
(7) Collagen adsorption and fibrosis treatment: immersing the RGD sequence covalent bond derived polysaccharide scaffold material into 3.0mg/mL collagen solution, immersing and incubating for 50h at 4 ℃, adjusting the collagen solution to pH=7 by using NaOH solution, immersing and washing for 12h by using PBS, and then freeze-drying to obtain the polysaccharide scaffold material, wherein the freezing temperature is as follows: -20 ℃, vacuum degree is: 1.00mbar, drying time 36h;
(8) And (3) sterilization: and carrying out double-layer sealing packaging and irradiation sterilization on the prepared polysaccharide stent material.
Comparative example 1
(1) Raw material selection: selecting fresh carrots;
(2) And (3) material treatment: cleaning radix Dauci Sativae, and peeling;
(3) And (3) material segmentation: cutting peeled radix Dauci Sativae into tissue blocks with thickness of 300 μm or less in longitudinal direction or transverse direction;
(4) Washing the cells with SDS: placing the tissue block in SDS solution (0.1%), oscillating for 12 hours at room temperature, replacing the new SDS solution, repeating for three times;
(5) Soaking and washing: immersing the tissue blocks washed by SDS into ultrapure water, shaking the tissue blocks by a shaking table for 24 hours, and replacing the ultrapure water, and repeating the steps for three times;
(6) PBS soaking: immersing the tissue blocks immersed and washed by pure water into PBS, shaking for 12 hours by using a shaking table, replacing the PBS, and repeating the process for three times to obtain a polysaccharide scaffold material;
(7) And (3) sterilization: and carrying out double-layer sealing packaging and irradiation sterilization on the prepared polysaccharide stent material.
Comparative example 2
(1) Raw material selection: selecting fresh carrots;
(2) And (3) material treatment: cleaning radix Dauci Sativae, and peeling;
(3) And (3) material segmentation: cutting peeled radix Dauci Sativae into tissue blocks with thickness of no more than 500 μm according to longitudinal direction or transverse direction;
(4) Washing the cells with SDS: placing the tissue block in SDS solution (0.1%), oscillating for 12 hours at room temperature, replacing the new SDS solution, repeating for three times;
(5) Soaking and washing: immersing the tissue blocks washed by SDS into ultrapure water, shaking the tissue blocks by a shaking table for 24 hours, and replacing the ultrapure water, and repeating the steps for three times;
(6) PBS soaking: immersing the tissue blocks rinsed by pure water into PBS, shaking the tissue blocks for 12 hours by using a shaking table, replacing the PBS, and repeating the steps for three times;
(7) Collagen coating treatment: immersing the tissue block soaked in the PBS in the step (6) into a collagen solution with the concentration of 1 mug/mL for incubation for 6 hours, and then immersing and cleaning the tissue block with the PBS for 3 hours to obtain a polysaccharide scaffold material;
(8) And (3) sterilization: and carrying out double-layer sealing packaging and irradiation sterilization on the prepared polysaccharide stent material.
Experimental example 1
The same as in example 3, except that fresh carrots were replaced with fresh peaches, white radishes, beef apples and apples, respectively, to prepare polysaccharide scaffold materials.
Cell culture
The results of cell culture of chicken muscle stem cells and chicken fat precursor cells were carried out using the polysaccharide scaffold materials prepared in examples 1 to 3, comparative examples 1 to 2 and experimental example 1, respectively, and are shown in tables 1 and 2.
Cell culture method:
Cryopreserved cells were resuscitated: taking out frozen cells from a cell bank, immediately placing the frozen cells into a water bath kettle at 37 ℃, thawing frozen solution, transferring the frozen cells into a centrifuge tube, adding 5mL of culture medium, blowing uniformly, centrifuging at 1000rpm for 5min, discarding the supernatant, adding the culture medium into the centrifuge tube, lightly blowing with a pipetting gun, transferring the resuspended cells into a culture dish, adding a proper amount of culture medium, lightly shaking the bottom of the culture dish to uniformly distribute the cells, and then placing the culture dish into an incubator at 37 ℃,5% CO 2 and saturated humidity for culture;
And (3) cell subculture expansion: changing a culture medium every two days in the cell culture process, digesting the cells until the cells are rounded and fall off by 0.25% pancreatin when the cells cover 80% of the area of the bottom of a culture dish, lightly blowing the cells until the cells fall off completely, adding enough culture medium to neutralize pancreatin, transferring the cells into a centrifuge tube, centrifuging at 1000rpm for 5min, discarding the supernatant, adding the culture medium into the centrifuge tube, lightly blowing the cells by a pipetting gun, re-suspending the cells, transferring the cells into the culture dish, adding a proper amount of culture medium, lightly shaking the bottom of the culture dish to ensure that the cells are uniformly distributed, and then placing the culture dish into a culture box with the temperature of 37 ℃, the concentration of CO 2 and the saturation humidity for culture;
Cell transfer flask culture: the polysaccharide scaffold material is placed in a culture medium and incubated for 12-24h (rotating speed of 30 rpm) in a rotating bottle system, after the cells are amplified and cultured to a sufficient quantity, all the cells are digested, collected and counted, and then the cells are inoculated into the rotating bottle culture system and cultured under the stirring condition of rotating speed of 30 rpm.
TABLE 1
TABLE 2
The rigid structure provided by the polysaccharide scaffold material prepared in examples 1-3 facilitates directional arrangement of muscle stem cells, and thus facilitates directional fusion of the muscle stem cells to form myotube cells, as shown in fig. 1;
The polysaccharide scaffold material prepared in the embodiment 1-3 has good biocompatibility, can be used for large-scale expansion culture of animal cells such as chicken muscle stem cells and chicken fat precursor cells, and has a cell culture density of 1X 10 7/mL as shown in the figure 1 and the figure 2, and the cell culture density is obviously better than the use effect of the comparative embodiment 1-2, as shown in the figure 3 and the figure 4.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (3)
1. The application of the polysaccharide scaffold material in cell culture is characterized in that the cells are chicken muscle stem cells or chicken fat precursor cells;
The preparation method of the polysaccharide scaffold material comprises the following steps:
freeze-drying plant tissues, and then soaking and washing the plant tissues with ultrapure water to obtain soaked and washed plant tissues;
Performing RGD sequence covalent bond derivatization and/or collagen adsorption fibrosis treatment on the plant tissues after infiltration and washing, and finally, performing freeze-drying to obtain the polysaccharide scaffold material;
The RGD sequence is Ac-Arg-Gly-Asp-Ser-Lys-NH 2;
In step (1), the conditions of the lyophilization process are: freezing temperature is-20 to-30 ℃, vacuum degree is 0.20-1.00 mbar, and drying time is 18-24 h;
in step (2), the treatment method for covalent bond derivatization of the RGD sequence comprises the following steps: in the presence of EDC, reacting the infiltrated and washed plant tissue with N-hydroxysuccinimide in a dimethylformamide solution, and then adding RGD sequence into the dimethylformamide solution for further reaction;
In step (2), the method of collagen adsorption fibrosis treatment comprises: immersing the plant tissues subjected to the immersion washing into a collagen solution for immersion incubation, then adjusting the collagen solution to be pH=7, and finally immersing and washing with PBS.
2. The use according to claim 1, wherein in step (1) the plant tissue is fresh vegetable and fruit tissue.
3. The use according to claim 1, wherein in step (2), the lyophilization conditions are: freezing temperature is-20 to-30 ℃, vacuum degree is 0.20-1.00 mbar, and drying time is 24-36 h.
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