CN114874975B - Method for culturing organoids by using elastin hydrogel - Google Patents
Method for culturing organoids by using elastin hydrogel Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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Abstract
The invention discloses a method for culturing organoids by using elastin hydrogel, which comprises the steps of dissolving soluble elastin in deionized water or buffer solution to prepare elastin aqueous solution; then adding acrylic ester-polyethylene glycol-N-hydroxysuccinimide or methacrylic ester-polyethylene glycol-N-hydroxysuccinimide to obtain modified elastin, dialyzing in deionized water, freezing, and freeze-drying to obtain dry modified elastin; dissolving the dry modified elastin in a phosphate physiological buffer solution, adding a photoinitiator, then mixing with organoid cell clusters, and irradiating under a light source with a certain wavelength to obtain an elastin hydrogel/cell complex; culturing in organoid culture medium, and amplifying cells in hydrogel to obtain organoid. The invention has simple process, rapid reaction, high elastin content and stable quality of the cultured organoids.
Description
Technical Field
The invention belongs to the field of biology, and particularly relates to a method for culturing organoids by using elastin hydrogel.
Background
Organoids are three-dimensional multicellular tissues that reproduce the (partial) structure and function of in vivo tissues formed by the self-assembly of stem cells or specific progenitor cells in the in vivo tissues in vitro (mainly epithelial cells). Organoids are therefore powerful models for studying tissue and organ development, wound repair, lesions, tumor tissue development, metastasis, drug resistance, targeted therapy, and immunotherapy. Current organoid cultures rely primarily on soluble basement membrane extracts of mouse sarcomas, such as commercially available matrigel. However, matrigel has the problem of undefined component content, which affects the quality of organoid culture. The compound is composed of a plurality of proteins (collagen, laminin and the like), polysaccharide and growth factors, and the components and the respective contents of each batch have larger differences, so that the cultured organoids have larger batch differences and have poorer stability. Thus, there is a need to develop organoid culture materials with defined compositions and levels.
Elastin (Elastin) is a water-insoluble three-dimensional network of soluble tropoelastin secreted by cells that is crosslinked by in vivo enzymes. Elastin exists in tissues and organs such as skin, lung, blood vessels, and the like, and plays a role in maintaining the elasticity of the tissues and organs. The elastin has the effects of promoting cell migration and proliferation and promoting vascularization, has the immune regulation function and good biocompatibility, and is an ideal cell culture material. There are two main classes of elastin-based biomaterials: the first is natural elastin extracted from animals, which has high yield and low cost, is suitable for large-scale production and application, but is insoluble in water and difficult to process, and is generally hydrolyzed into soluble elastin for application; the second is to make bacteria synthesize human tropoelastin or elastin-like polypeptide by genetic engineering method, but the method has low yield and high cost. Therefore, if the hydrolyzed soluble natural elastin is modified, the hydrogel material which can be formed in situ is prepared, and the hydrogel material is expected to be applied to organoid culture.
However, the studies (such as Polymers,2020,12,670) on the preparation of hydrogels by modifying natural elastin in the prior literature have problems including multiple (two) pre-reaction steps, long time (the first reaction step takes 1 hour, the second reaction step takes overnight), low efficiency (mercapto groups are easy to cross-link in molecules to affect the subsequent grafting reaction, and mercapto groups can react with double bonds at both ends of polyethylene glycol diacrylate at the same time to cause the polyethylene glycol diacrylate grafted on elastin to lose photocrosslinking ability); the molecular weight of polyethylene glycol diacrylate used in the reaction is large, so that the protein content in the final product is only about 60%, and the high polyethylene glycol content can impair the cell adhesion of the hydrogel; in addition, the grafting rate in the reaction is only about 60% (the change of primary amino group content before and after modification is measured). Thus, there is an urgent need to improve the modification process of elastin hydrogels, which in turn allow for the cultivation of stable organoids.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a method for culturing organoids by using elastin hydrogel, which has good stability and high efficiency.
The aim of the invention is achieved by the following technical scheme:
a method for culturing organoids using elastin hydrogels, comprising the steps of:
(1) Dissolving 0.5-2 parts by mass of soluble elastin in 100 parts of deionized water or buffer solution to prepare elastin aqueous solution;
(2) Adding 0.1-1 part by mass of acrylic ester-polyethylene glycol-N-hydroxysuccinimide (AC-PEG-NHS) or methacrylic ester-polyethylene glycol-N-hydroxysuccinimide (MeAC-PEG-NHS) into the elastin aqueous solution, and carrying out reaction under the condition of nitrogen filling and light shielding at normal temperature to obtain modified elastin;
(3) Dialyzing the modified elastin in deionized water, putting the dialyzed modified elastin aqueous solution in an environment of < -15 ℃ to freeze the dialyzed modified elastin aqueous solution, and then freeze-drying the dialyzed modified elastin aqueous solution in a freeze dryer to obtain dry modified elastin;
(4) Dissolving 0.5-20 parts by mass of the dry modified elastin in 100 parts by mass of phosphate Physiological (PBS) buffer solution, and adding a photoinitiator to obtain elastin hydrogel precursor solution;
(5) Digesting the tissue or tumor tissue into single-cell suspension, performing cell counting, and centrifuging to obtain a cell mass; mixing the elastin hydrogel precursor solution with a cell mass, and then irradiating under a light source with a certain wavelength to obtain an elastin hydrogel/cell complex; culturing the elastin hydrogel/cell complex in an organoid medium; under the action of the culture medium and the hydrogel, epithelial cells in the cells wrapped by the hydrogel can be amplified in the hydrogel to form organoids.
Preferably, in step (1), the soluble elastin is derived from a hydrolysate of insoluble elastin in fish or mammalian tissue.
Preferably, in the step (1), the buffer solution is a phosphate physiological buffer solution with the concentration of 0.01M or a NaH 2PO4 buffer solution with the concentration of 0.01-0.06M; the pH value of the buffer solution is 8.0-8.3.
Preferably, in step (2), the degree of polymerization of PEG in AC-PEG-NHS or MeAC-PEG-NHS is 4-20.
Preferably, in step (2), the reaction time of AC-PEG-NHS or MeAC-PEG-NHS with the aqueous elastin solution is 0.5 to 3 hours.
Preferably, in the step (3), the dialysis time of the modified elastin is 24-48 hours, and the molecular weight cut-off of the dialysis bag is 1-50 kDa.
Preferably, in step (4), the concentration of the phosphate physiological buffer solution is 0.01M and the pH value is 7.0-7.5.
Preferably, in the step (4), the photoinitiator is an ultraviolet photoinitiator or a blue photoinitiator; the ultraviolet initiator can be 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylpropionne (Irgacure 2959); the blue photoinitiator may be phenyl-2, 4, 6-trimethylbenzoyl lithium phosphinate (LAP); the final concentration of the added initiator is 0.01-0.5%.
In the step (4), 0.1 to 10 parts by mass of other water-soluble polymers with photocrosslinking functional groups can be added, and the water-soluble polymers and 0.5 to 10 parts by mass of the dry modified elastin are dissolved in 100 parts of phosphate physiological buffer solution, and then 0.01 to 0.5 part of photoinitiator is added to form the composite hydrogel precursor.
Other water-soluble polymers with photocrosslinking functionality include, but are not limited to, the original or modified products of the following polymers: protein polymers (gelatin, collagen, silk fibroin, polypeptides, keratin), polysaccharide polymers (alginic acid, hyaluronic acid, chitosan, gellan gum, carrageenan), other synthetic water-soluble polymers, or any combination of the above polymers.
Preferably, in the step (5), the wavelength of the irradiation light is 365nm or 405nm, and the photo-crosslinking irradiation time is 0.5 to 10 minutes.
Preferably, in step (5), the concentration of cells in the modified elastin hydrogel precursor solution is 0.5-5x10 6 cells/mL.
In step (5), the single cell suspension includes cells isolated from normal tissue, or cells isolated from tumor tissue, and also includes cells commercially available for organoid formation.
In the step (5), the mixing temperature of the organoid cell mass and the elastin hydrogel precursor solution is 4-37 ℃.
The principle of the invention is as follows: (1) The acrylate-polyethylene glycol-N-hydroxysuccinimide (AC-PEG-NHS) or methacrylate-polyethylene glycol-N-hydroxysuccinimide (MeAC-PEG-NHS) is used to react with elastin and graft double bonds which can undergo photo-crosslinking reaction to elastin. The AC-PEG-NHS or MeAC-PEG-NHS adopted by the invention can directly react with primary amine on elastin in a nucleophilic way by the hydroxysuccinimide ester group during the reaction, thereby grafting double bonds on primary amine of elastin. The modified elastin prepared by the invention contains double bonds, and can mutually perform addition reaction under the irradiation of a photocatalyst and a light source with a certain wavelength to form the three-dimensional crosslinked polymer network-hydrogel. (2) When isolated cells from tissue or tumor tissue are cultured in elastin hydrogels, the epithelial cells selectively grow rapidly while other cell types proliferate slower or do not grow under the action of the medium. Thus, over time, the cells within the hydrogel will be predominantly epithelial. And elastin hydrogels provide an environment similar to the three-dimensional extracellular matrix microenvironment in vivo tissue, helping the epithelial cells encapsulated within the hydrogel spontaneously form structures therein that reproduce in vivo tissue, thus forming stable organoid structures.
Compared with the prior art, the invention has the following advantages and effects:
(1) The preparation method of the photo-crosslinked elastin hydrogel precursor, namely the modified elastin, only needs one step, and the method in the prior art needs two steps, so that the method is simpler.
(2) The modification reaction time of the elastin grafted double bond can be controlled to be 0.5-3 hours, and the reaction of the elastin grafted double bond (the reaction with polyethylene glycol diacrylate) in the prior art needs to be carried out overnight, so that the reaction time is longer, and the preparation efficiency of the method is high.
(3) The grafting efficiency of primary amino groups on the elastin can reach more than 90%, and the grafting efficiency of the prior art on the primary amino groups on the elastin is only about 60%, so that the method disclosed by the invention is high in modified grafting efficiency.
(4) The PEG polymerization degree in the graft acrylate-polyethylene glycol-N-hydroxysuccinimide (AC-PEG-NHS) or methacrylate-polyethylene glycol-N-hydroxysuccinimide (MeAC-PEG-NHS) used in the invention can be controlled to be 1-20, and the molecular weight can be controlled to be 300-1300Da; whereas the molecular weight of the polyethylene glycol diacrylate grafted in the prior art is 8000; therefore, the protein content of the modified final product of the invention can reach more than 90 percent, and the protein content of the modified final product in the prior art is about 60 percent at most; because polyethylene glycol has anti-adhesion property, the improvement of the protein content in the hydrogel helps to improve the cell adhesion of the hydrogel.
(5) The elastin hydrogel precursor solution prepared by the method can be independently used for wrapping cell culture organoids, but the prior art only prepares elastin/gelatin/polyethylene glycol composite hydrogel for wrapping cells, and does not prepare pure modified elastin hydrogel for wrapping cells.
(6) In the invention, organoid cells can be mixed with elastin hydrogel at room temperature, and then light irradiation crosslinking is carried out to obtain hydrogel/cell compound, and the reaction condition is mild; in the prior art, the matrigel and the cells must be mixed at about 4 degrees to avoid premature gelation of the matrigel, and the reaction conditions are severe.
(7) In the invention, the elastin hydrogel or the hydrogel formed by the elastin hydrogel and other photocrosslinkable polymers has definite components and does not contain growth factors, so that the quality stability of cultured organoids can be ensured; the major problems of the prior art are large batch differences in the components of the matrigel, including major protein components and contents, and major growth factor components and contents, which result in batch quality differences in cultured organoids.
Drawings
FIG. 1 is a schematic reaction scheme of AC-PEG-NHS modified elastin.
FIG. 2 is a Fourier transform infrared spectrum (FTIR) of unmodified Elastin (Elastin) and modified Elastin (Elastin-PEG-AC) prepared in example 9.
FIG. 3 is an organoid (scale: 200 μm.) of test example 1 cultured for 7 days with an elastin/gelatin composite hydrogel.
FIG. 4 is a graph showing organoids (scale: 200 μm.) cultured for 7 days with matrigel in comparative example 1.
Detailed Description
In order that the invention may be readily understood, a detailed description of the invention will be provided below with reference to specific examples. The following examples will assist those skilled in the art in further understanding the present invention, but are not intended to limit the invention in any way. It should be noted that the present invention can be modified and improved by those skilled in the art without departing from the spirit of the present invention, which falls within the scope of the present invention.
Example 1
The embodiment provides a modification method of photo-crosslinkable elastin, which comprises the following steps: 0.05g of soluble elastin was dissolved in 10mL of 0.01M phosphate physiological buffer pH 8.0 to prepare an aqueous elastin solution. To the above elastin aqueous solution, 0.01g of methacrylate-polyethylene glycol-N-hydroxysuccinimide (polymerization degree of polyethylene glycol: 4) was added, and the mixture was stirred at room temperature under nitrogen and in a dark place for 3 hours. The reaction was dialyzed in a dialysis bag with a molecular weight cut-off of 3kDa for 48 hours, and then the purified protein solution was lyophilized in a lyophilizer to obtain the modified elastin.
Example 2
The embodiment provides a modification method of photo-crosslinkable elastin, which comprises the following steps: 0.2g of soluble elastin was dissolved in 10mL of 0.01M phosphate physiological buffer pH 8.0 to prepare an aqueous elastin solution. To the above elastin aqueous solution, 0.1g of methacrylate-polyethylene glycol-N-hydroxysuccinimide (polyethylene glycol polymerization degree 20) was added, and the mixture was stirred at room temperature under nitrogen and in a dark place for 0.5 hours. The reaction was dialyzed in a dialysis bag with a molecular weight cut-off of 3kDa for 48 hours, and then the purified protein solution was lyophilized in a lyophilizer to obtain the modified elastin.
Example 3
The embodiment provides a modification method of photo-crosslinkable elastin, which comprises the following steps: 0.15g of soluble elastin was dissolved in 10mL of 0.01M phosphate physiological buffer pH 8.3 to prepare an aqueous elastin solution. To the above elastin aqueous solution, 0.08g of methacrylate-polyethylene glycol-N-hydroxysuccinimide (polymerization degree of polyethylene glycol 10) was added, and the mixture was stirred at room temperature under nitrogen and in a dark place for 2 hours. The reaction was dialyzed in a dialysis bag with a molecular weight cut-off of 3kDa for 48 hours, and then the purified protein solution was lyophilized in a lyophilizer to obtain the modified elastin.
Example 4
The embodiment provides a modification method of photo-crosslinkable elastin, which comprises the following steps: 0.05g of soluble elastin was dissolved in 10mL of 0.01M NaH 2PO4 buffer pH 8.0 to prepare an aqueous elastin solution. To the above elastin aqueous solution, 0.01g of methacrylate-polyethylene glycol-N-hydroxysuccinimide (polymerization degree of polyethylene glycol: 8) was added, and the mixture was stirred at room temperature under nitrogen and in a dark place for 1 hour. The reaction was dialyzed in a dialysis bag with a molecular weight cut-off of 3kDa for 48 hours, and then the purified protein solution was lyophilized in a lyophilizer to obtain the modified elastin.
Example 5
The embodiment provides a modification method of photo-crosslinkable elastin, which comprises the following steps: 0.15g of soluble elastin was dissolved in 10mL of 0.01M NaH 2PO4 buffer pH 8.3 to prepare an aqueous elastin solution. To the above elastin aqueous solution, 0.05g of methacrylate-polyethylene glycol-N-hydroxysuccinimide (polymerization degree of polyethylene glycol 10) was added, and the mixture was stirred at room temperature under nitrogen and in a dark place for 3 hours. The reaction was dialyzed in a dialysis bag with a molecular weight cut-off of 3kDa for 48 hours, and then the purified protein solution was lyophilized in a lyophilizer to obtain the modified elastin.
Example 6
The embodiment provides a modification method of photo-crosslinkable elastin, which comprises the following steps: 0.2g of soluble elastin was dissolved in 10mL of 0.06M NaH 2PO4 buffer pH 8.3 to prepare an aqueous elastin solution. To the above elastin aqueous solution, 0.1g of methacrylate-polyethylene glycol-N-hydroxysuccinimide (polymerization degree of polyethylene glycol: 4) was added, and the mixture was stirred at room temperature under nitrogen and in a dark place for 0.5 hours. The reaction was dialyzed in a dialysis bag with a molecular weight cut-off of 3kDa for 48 hours, and then the purified protein solution was lyophilized in a lyophilizer to obtain the modified elastin.
Example 7
The embodiment provides a modification method of photo-crosslinkable elastin, which comprises the following steps: 0.08g of soluble elastin was dissolved in 10mL of 0.01M phosphate physiological buffer pH 8.0 to prepare an aqueous elastin solution. To the above elastin aqueous solution was added 0.02g of acrylate-polyethylene glycol-N-hydroxysuccinimide (polymerization degree of polyethylene glycol 4), and the mixture was stirred at room temperature under nitrogen in the absence of light for 3 hours. The reaction was dialyzed in a dialysis bag with a molecular weight cut-off of 3kDa for 48 hours, and then the purified protein solution was lyophilized in a lyophilizer to obtain the modified elastin. The reaction scheme of the AC-PEG-NHS modified elastin is shown in FIG. 1.
Example 8
The embodiment provides a modification method of photo-crosslinkable elastin, which comprises the following steps: 0.15g of soluble elastin was dissolved in 10mL of 0.01M phosphate physiological buffer pH 8.3 to prepare an aqueous elastin solution. To the above elastin aqueous solution, 0.1g of acrylate-polyethylene glycol-N-hydroxysuccinimide (polyethylene glycol polymerization degree 20) was added, and the mixture was stirred at room temperature under nitrogen in the absence of light for 0.5 hour. The reaction was dialyzed in a dialysis bag with a molecular weight cut-off of 3kDa for 48 hours, and then the purified protein solution was lyophilized in a lyophilizer to obtain the modified elastin.
Example 9
The embodiment provides a modification method of photo-crosslinkable elastin, which comprises the following steps: 0.1g of soluble elastin was dissolved in 10mL of 0.06M NaH 2PO4 buffer pH 8.3 to prepare an aqueous elastin solution. To the above elastin aqueous solution, 0.04g of acrylic ester-polyethylene glycol-N-hydroxysuccinimide (polymerization degree of polyethylene glycol: 4) was added, and the mixture was stirred at room temperature under nitrogen and under dark conditions for 3 hours. The reaction was dialyzed in a dialysis bag with a molecular weight cut-off of 3kDa for 48 hours, and then the purified protein solution was lyophilized in a lyophilizer to obtain the modified elastin.
The modified elastin prepared in any of examples 1-9 was characterized using a fourier transform infrared spectrometer. The modified elastin can be found to have new absorption peaks at 892 and 1069 wave numbers, namely the out-of-plane bending vibration of C-H of carbon-carbon double bond on acrylic acid and the C-O-C telescopic vibration absorption peak on polyethylene glycol (PEG), which prove that the modification is successful, as shown in figure 2.
Example 10
The present example provides a method for culturing a gastric organoid using a photo-crosslinkable elastin hydrogel: in examples 1-9, 50mg of the lyophilized modified elastin prepared in any of examples was dissolved in 2mL of a phosphate physiological buffer solution (pH 7.4) containing the blue photoinitiator lithium phenyl-2, 4, 6-trimethylbenzoyl phosphinate (0.5 mg/mL) to prepare a 25mg/mL elastin hydrogel precursor solution. Through approval by the ethical committee, normal stomach tissue discarded from surgery was removed and digested into a single cell suspension. Cell clusters were prepared by centrifugation of a suspension of 2x10 6 cells. The cell pellet was mixed with sterile filtered elastin hydrogel precursor solution in a biosafety cabinet at 10 ℃ (1 x10 6 cells/mL). Next, 40uL of the mixture was dropped into the wells of the plate by a pipette, and after irradiation with a blue light meter (wavelength 405 nm) for 2 minutes, hydrogel/cell complexes were formed in situ. Organoid medium is then added to the wells, and the cells gradually expand within the hydrogel and form the gastric organoids.
Example 11
The present example provides a method for culturing a gastric organoid using a photo-crosslinkable elastin hydrogel: in examples 1-9, 200mg of the lyophilized modified elastin prepared in any of examples was dissolved in 2mL of a phosphate physiological buffer solution (pH 7.4) containing the blue photoinitiator lithium phenyl-2, 4, 6-trimethylbenzoyl phosphinate (5 mg/mL) to prepare a 100mg/mL elastin hydrogel precursor solution. Through approval by the ethical committee, normal stomach tissue discarded from surgery was removed and digested into a single cell suspension. Cell clusters were prepared by centrifugation of a suspension of 4x10 6 cells. The cell pellet was mixed with sterile filtered elastin hydrogel precursor solution in a biosafety cabinet at room temperature (2 x10 6 cells/mL). Next, 40uL of the mixture was dropped into the wells of the plate by a pipette, and after 1 minute of irradiation with a blue light meter (wavelength 405 nm), hydrogel/cell complexes were formed in situ. Organoid medium is then added to the wells, and the cells gradually expand within the hydrogel and form the gastric organoids.
Example 12
The present example provides a method for culturing gastric cancer organoids using a photo-crosslinkable elastin hydrogel: : in examples 1-9, 100mg of the lyophilized modified elastin prepared in any of examples was dissolved in 2mL of a phosphate physiological buffer solution (pH 7.4) containing the blue photoinitiator lithium phenyl-2, 4, 6-trimethylbenzoyl phosphinate (2 mg/mL) to prepare a 50mg/mL elastin hydrogel precursor solution. Through examination and approval by the ethical committee, gastric cancer tumor tissue obtained during surgery was taken and digested into single cell suspensions. The cell mass was prepared by centrifuging a suspension of 3x10 6 cells. The cell pellet was mixed with sterile filtered elastin hydrogel precursor solution in a biosafety cabinet at room temperature (1.5x10 6 cells/mL). Subsequently, 30uL of the mixture was dropped into wells of a culture plate by a pipette, and after irradiation with a blue light (wavelength 405 nm) for 0.5 minutes, a hydrogel/cell complex was formed in situ. Then organoid medium is added to the wells, and the cells gradually expand within the hydrogel and form gastric carcinoma organoids.
Example 13
The present example provides a method for culturing colorectal cancer organoids using a photo-crosslinkable elastin hydrogel: in examples 1-9, 150mg of the lyophilized modified elastin prepared in any of examples was dissolved in 2mL of a phosphate physiological buffer solution (pH 7.4) containing the ultraviolet initiator 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylpropionne (1 mg/mL) to prepare a 75mg/mL elastin hydrogel precursor solution. The ethical committee approved that the tumor tissue of the rectum cancer obtained in the operation was taken and digested into single cell suspension. Cell clusters were prepared by centrifugation of a 1x10 7 cell suspension. The cell pellet was mixed with sterile filtered elastin hydrogel precursor solution in a biosafety cabinet at room temperature (5 x10 6 cells/mL). Next, 20uL of the mixture was dropped into the wells of the culture plate by a pipette, and after irradiation with an ultraviolet (wavelength 365 nm) for 1.5 minutes, a hydrogel/cell complex was formed in situ. Organoid medium is then added to the wells, and the cells gradually expand within the hydrogel and form colorectal organoids.
Example 14
The present example provides a method for culturing a gastric organoid using a photo-crosslinkable elastin/gelatin composite hydrogel: in examples 1-9, 100mg of the lyophilized modified elastin prepared in any of examples was dissolved in 2mL of a phosphate physiological buffer solution (pH 7.4) containing the ultraviolet initiator 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylpropionbenzene (0.5 mg/mL) to prepare a 50mg/mL elastin hydrogel precursor solution. 50mg of the methacrylated gelatin is added into the precursor solution and dissolved, so as to prepare 50mg/mL of composite hydrogel precursor solution. Through approval by the ethical committee, normal stomach tissue discarded from surgery was removed and digested into a single cell suspension. Cell clusters were prepared by centrifugation of a suspension of 5x10 6 cells. The cell pellet was mixed with sterile filtered complex protein hydrogel precursor solution in a biosafety cabinet at room temperature (2.5x10 6 cells/mL). Next, 40uL of the mixture was dropped into the wells of the plate by a pipette, and after irradiation with an ultraviolet (wavelength 365 nm) for 2 minutes, a hydrogel/cell complex was formed in situ. Organoid medium is then added to the wells, and the cells gradually expand within the hydrogel and form the gastric organoids.
Example 15
The present example provides a method for culturing colorectal cancer organoids using a photo-crosslinkable elastin/hyaluronic acid composite hydrogel: in examples 1-9, 50mg of the lyophilized modified elastin prepared in any of examples was dissolved in 2mL of a phosphate physiological buffer solution (pH 7.4) containing the blue photoinitiator lithium phenyl-2, 4, 6-trimethylbenzoyl phosphinate (1 mg/mL) to prepare 25mg/mL of an elastin hydrogel precursor solution. To this precursor solution, 20mg of methacrylated hyaluronic acid was added and dissolved to prepare 35mg/mL of a composite hydrogel precursor solution. Through examination and approval by the ethical committee, intestinal cancer tumor tissue obtained during surgery was taken and digested into single cell suspensions. Cell clusters were prepared by centrifugation of a suspension of 4x10 6 cells. The cell pellet was mixed with sterile filtered complex protein hydrogel precursor solution in a biosafety cabinet at room temperature (2 x10 6 cells/mL). Next, 40uL of the mixture was dropped into the wells of the plate by a pipette, and after irradiation with a blue light meter (wavelength 405 nm) for 2 minutes, hydrogel/cell complexes were formed in situ. Organoid medium is then added to the wells, and the cells gradually expand within the hydrogel and form colorectal organoids.
Example 16
The present example provides a method for culturing gastric cancer organoids using a photo-crosslinkable elastin/gelatin composite hydrogel: in examples 1-9, 40mg of the lyophilized modified elastin prepared in any of examples was dissolved in 2mL of a phosphate physiological buffer solution (pH 7.4) containing the blue photoinitiator lithium phenyl-2, 4, 6-trimethylbenzoyl phosphinate (1 mg/mL) to prepare a 20mg/mL elastin hydrogel precursor solution. 40mg of methacrylate gelatin was added to the precursor solution and dissolved to prepare 40mg/mL of a composite hydrogel precursor solution. Stomach cancer tumor tissue obtained in operation is taken and digested into single cell suspension after passing examination by the corresponding ethical committee. The cell mass was prepared by centrifuging a suspension of 3x10 6 cells. The cell pellet was mixed with sterile filtered complex protein hydrogel precursor solution in a biosafety cabinet at room temperature (1.5x10 6 cells/mL). Next, 40uL of the mixture was dropped into the wells of the plate by a pipette, and after 1 minute of irradiation with a blue light meter (wavelength 405 nm), hydrogel/cell complexes were formed in situ. Then organoid medium is added to the wells, and the cells gradually expand within the hydrogel and form gastric carcinoma organoids.
Test example 1
The culture of elastin/gelatin composite hydrogel in gastric cancer organoids is used as a test example. In examples 1 to 9, 50mg of the lyophilized modified elastin prepared in any of examples was dissolved in 2mL of a phosphate physiological buffer solution (pH 7.4) containing lithium (2 mg/mL) of blue-light initiator phenyl-2, 4, 6-trimethylbenzoyl phosphinate to prepare 25mg/mL of elastin hydrogel precursor solution. 50mg of the methacrylated gelatin is added into the precursor solution and dissolved, so as to prepare 50mg/mL of composite hydrogel precursor solution. Through approval by the ethical committee, gastric cancer tissue obtained during surgery was taken and digested into single cell suspensions. Cell clusters were prepared by centrifugation of a suspension of 4x10 6 cells. The cell pellet was mixed with sterile filtered complex protein hydrogel precursor solution in a biosafety cabinet at room temperature (2 x10 6 cells/mL). Next, 40uL of the mixture was dropped into the wells of the plate by a pipette, and after 1 minute of irradiation with a blue light meter (wavelength 405 nm), hydrogel/cell complexes were formed in situ. Then organoid medium is added to the wells, and the cells gradually expand within the hydrogel and form gastric carcinoma organoids. Organoid formation was observed after 1,3, 7 days of culture, and it was found that the hydrogel can support organoid formation and growth, and the size of gastric cancer organoids formed was uniform, as shown in fig. 3. Therefore, the organoids cultured by the method have stable quality and small variability.
Comparative example 1
Through approval by the ethical committee, gastric cancer tissue obtained during surgery was taken and digested into single cell suspensions. Cell clusters were prepared by centrifugation of a suspension of 0.5x10 6 cells. The cell pellet was mixed with 0.25mL matrigel at 4 ℃ in a biosafety cabinet (2 x10 6 cells/mL). Subsequently, 40uL of the mixture was dropped into wells of a culture plate by a pipette, and the mixture was placed in an incubator at 37℃for 5 minutes to gel the mixture. Then organoid specific medium is added to the wells and the cells will gradually expand within the matrigel and form gastric cancer organoids. Organoids were observed after 1, 3, and 7 days of culture, and matrigel was found to support organoid formation and growth, but the size of gastric cancer organoids formed was very uneven, as shown in fig. 4.
The foregoing is illustrative of the present invention, and the present invention is not limited to the above embodiments, but is capable of other modifications, adaptations, alternatives, combinations, and simplifications without departing from the spirit and principles of the invention.
Claims (5)
1. A method for culturing gastric cancer organoids by using elastin hydrogel, comprising the steps of:
(1) Dissolving 0.5-2 parts by mass of soluble elastin in 100 parts of deionized water or buffer solution to prepare an elastin aqueous solution;
(2) Adding 0.1-1 part by mass of acrylic ester-polyethylene glycol-N-hydroxysuccinimide or methacrylic ester-polyethylene glycol-N-hydroxysuccinimide into the elastin aqueous solution, and carrying out reaction under the condition of nitrogen charging and light shielding at normal temperature to obtain modified elastin; the polymerization degree of polyethylene glycol in the acrylic ester-polyethylene glycol-N-hydroxysuccinimide or the methacrylic ester-polyethylene glycol-N-hydroxysuccinimide is 4-20;
(3) Dialyzing the modified elastin in deionized water, putting the dialyzed modified elastin aqueous solution in an environment of < -15 ℃ to freeze the dialyzed modified elastin aqueous solution, and then freeze-drying the dialyzed modified elastin aqueous solution in a freeze dryer to obtain dry modified elastin;
(4) Dissolving 0.5-20 parts by mass of dry modified elastin in 100 parts of phosphate physiological buffer solution, and adding a photoinitiator to prepare elastin hydrogel precursor solution; then adding methacrylate gelatin into the precursor solution and dissolving to obtain a composite hydrogel precursor solution;
(5) Digesting stomach cancer tissues into single-cell suspension, performing cell counting, and centrifuging to obtain cell clusters; mixing the composite hydrogel precursor solution with a cell mass, and then irradiating under a light source to obtain a hydrogel-cell composite; culturing the hydrogel-cell complex in a gastric cancer organoid culture medium; under the action of the culture medium and the hydrogel, epithelial cells in the cells wrapped by the hydrogel can be amplified in the hydrogel to form gastric cancer organoids.
2. The method for culturing gastric cancer organoids using elastin hydrogels of claim 1, wherein: in step (1), the soluble elastin is derived from a hydrolysate of insoluble elastin in fish or mammalian tissue; the buffer solution is 0.01M phosphate physiological buffer solution or 0.01-0.06M NaH 2PO4 buffer solution; the pH value of the buffer solution is 8.0-8.3.
3. The method for culturing gastric cancer organoids using elastin hydrogels of claim 1, wherein: in the step (2), the reaction time of the acrylic ester-polyethylene glycol-N-hydroxysuccinimide or the methacrylic ester-polyethylene glycol-N-hydroxysuccinimide and the elastin aqueous solution is 0.5-3 hours.
4. The method for culturing gastric cancer organoids using elastin hydrogels of claim 1, wherein: in the step (4), the photoinitiator is an ultraviolet initiator or a blue photoinitiator; the ultraviolet initiator is 2-hydroxy-4' - (2-hydroxyethoxy) -2-methyl propiophenone; the blue photoinitiator is phenyl-2, 4, 6-trimethyl benzoyl lithium phosphinate; the final concentration of the added initiator is 0.01% -0.5%.
5. The method for culturing gastric cancer organoids using elastin hydrogels of claim 1, wherein: in the step (5), the wavelength of the irradiation light source is 365 nm or 405 nm, and the irradiation time is 0.5-10 minutes.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20090120504A (en) * | 2007-03-07 | 2009-11-24 | 에코 테라퓨틱스, 인크. | Transdermal analyte monitoring system and method for analyte detection |
| CN105431179A (en) * | 2014-08-15 | 2016-03-23 | 苏州堪赛尔生物技术有限公司 | Matrix scaffold for three-dimensional cell culturing and construction method and use thereof |
| CN110272860A (en) * | 2019-06-30 | 2019-09-24 | 东南大学苏州医疗器械研究院 | A kind of three-dimensional cell cultivation microenvironment construction method and application |
| CN111936560A (en) * | 2018-02-28 | 2020-11-13 | 波尔多大学 | Hydrogel for stimulating nerve regeneration, osteogenesis and angiogenesis |
| CN111962210A (en) * | 2020-06-22 | 2020-11-20 | 华南理工大学 | Polycaprolactone/methacryloylated elastin nanofiber composite membrane and preparation method and application thereof |
| CN113215102A (en) * | 2021-04-01 | 2021-08-06 | 南京医科大学 | Culture medium and culture method for organoid |
| CN114133435A (en) * | 2021-07-21 | 2022-03-04 | 浙江大学 | Elastin-like polypeptide and application |
| CN114231490A (en) * | 2021-12-23 | 2022-03-25 | 北京基石生命科技有限公司 | Method for culturing tumor organoid by permeable hydrogel scaffold |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9725498B2 (en) * | 2009-07-17 | 2017-08-08 | The Texas A&M University System | Designer collagens and use thereof |
| US11406737B2 (en) * | 2017-08-22 | 2022-08-09 | Northeastern University | Gelatin/elastin composites for peripheral nerve repair |
-
2022
- 2022-05-09 CN CN202210499600.1A patent/CN114874975B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20090120504A (en) * | 2007-03-07 | 2009-11-24 | 에코 테라퓨틱스, 인크. | Transdermal analyte monitoring system and method for analyte detection |
| CN105431179A (en) * | 2014-08-15 | 2016-03-23 | 苏州堪赛尔生物技术有限公司 | Matrix scaffold for three-dimensional cell culturing and construction method and use thereof |
| CN111936560A (en) * | 2018-02-28 | 2020-11-13 | 波尔多大学 | Hydrogel for stimulating nerve regeneration, osteogenesis and angiogenesis |
| CN110272860A (en) * | 2019-06-30 | 2019-09-24 | 东南大学苏州医疗器械研究院 | A kind of three-dimensional cell cultivation microenvironment construction method and application |
| CN111962210A (en) * | 2020-06-22 | 2020-11-20 | 华南理工大学 | Polycaprolactone/methacryloylated elastin nanofiber composite membrane and preparation method and application thereof |
| CN113215102A (en) * | 2021-04-01 | 2021-08-06 | 南京医科大学 | Culture medium and culture method for organoid |
| CN114133435A (en) * | 2021-07-21 | 2022-03-04 | 浙江大学 | Elastin-like polypeptide and application |
| CN114231490A (en) * | 2021-12-23 | 2022-03-25 | 北京基石生命科技有限公司 | Method for culturing tumor organoid by permeable hydrogel scaffold |
Non-Patent Citations (4)
| Title |
|---|
| Bioactive hydrogels based on Designer Collagens;E. Cosgriff-Hernandez等;Acta Biomaterialia;第6卷(第10期);摘要、第3968-3977页 * |
| Inclusion of Cross-Linked Elastin in Gelatin/PEG Hydrogels Favourably Influences Fibroblast Phenotype;Ye Cao等;Polymers(Basel);第12卷(第3期);摘要、第2.1-2.2、2.4-2.5、3.1-3.3节 * |
| Incorporation of adhesion peptides into nonadhesive hydrogels useful for tissue resurfacing;Diane L.Hern 等;Journal of biomaterials research;第39卷(第2期);摘要、第266页左栏第1段-第275页右栏第1段 * |
| Injectable Laminin-Functionalized Hydrogel for Nucleus Pulposus Regeneration;Aubrey T. Francisco等;Biomaterials;第34卷(第30期);摘要、第2-10页 * |
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