CN114908034A - Acellular fluid, acellular matrix hydrogel and preparation method and application thereof - Google Patents

Acellular fluid, acellular matrix hydrogel and preparation method and application thereof Download PDF

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CN114908034A
CN114908034A CN202210458274.XA CN202210458274A CN114908034A CN 114908034 A CN114908034 A CN 114908034A CN 202210458274 A CN202210458274 A CN 202210458274A CN 114908034 A CN114908034 A CN 114908034A
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廖传荣
朱宇
宋志文
黄敏
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Accurate International Biotechnology Guangzhou Co ltd
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Abstract

The invention relates to a cell removal solution, a cell removal matrix hydrogel and a preparation method and application thereof, wherein the cell removal solution comprises the following components in final concentration: NaCl or NH 4 Cl, 1-5 mol/L; 1-10% of sodium deoxycholate; DMSO, 0.5-5%; 0.1 to 5 percent of tributyl phosphate; 0.01-0.5mol/L of glycine; the solvent is water. The preparation method of the acellular matrix hydrogel comprises the following steps: pre-treating the collected tissue blocks; shearing the tissue blocks obtained in the step 1, and then placing the sheared tissue blocks in the cell removing liquid to be stirred to obtain a cell removing scaffold; washing the decellularized scaffoldFreeze-drying, grinding into matrix powder; digesting the matrix powder with a digestive fluid to obtain a pre-gel; filtering out undigested impurities in the pre-gel, and then adjusting the pH value to 6.5-7.5 to obtain the product. The acellular matrix hydrogel disclosed by the invention is used for organoid culture, has good biocompatibility with organoid tissues and cells, has a good tissue repair effect, can be better compatible with the cells, and is favorable for survival and proliferation of the cells cultured by 3D.

Description

Acellular fluid, acellular matrix hydrogel and preparation method and application thereof
Technical Field
The invention relates to the technical field of biological materials, in particular to a cell-free solution, a cell-free matrix hydrogel, and a preparation method and application thereof.
Background
Organoids are a three-dimensional cell culture technique that can mimic the tissue structure and gene expression of organs in vivo in vitro. Organoids are three-dimensional aggregates formed by self-differentiation and self-assembly of various cells including stem cells in vitro, and the cell types, the cell proportions, the spatial structures and the arrangement of the cells have higher similarity with those of internal organs, and can simulate partial functions of the internal organs. Furthermore, organoid gene expression is much closer to that in vivo than in conventional two-dimensional cell culture. Therefore, organoid-based biological activity assays are more relevant in vivo. Therefore, organoids are currently widely used in biological research, pharmaceutical research, medical research, precision medicine transformation applications, and the like.
Currently, organoid culture is mainly supported and spaced by acellular matrix hydrogels from biomaterial sources. The acellular matrix hydrogel can better retain extracellular matrix protein components, active factors and natural three-dimensional structures of original tissues or organs, and has more advantages in the aspects of maintaining cell characteristics and subsequent regenerative medicine compared with artificially synthesized materials and natural single-component materials. The tissue repairing agent can well restore the microenvironment before tissue damage, has the biological activity of promoting cell adhesion, proliferation and differentiation and the characteristic of biodegradability, and has more and more reports to prove that the biological function and the repairing effect have tissue specificity. However, the preparation of acellular matrix hydrogel is mainly based on physical or chemical methods, and at present, no unified and standardized method exists, so that the operation is complicated, the acellular degree is low, and cell residues are easy to remain. The physical method is generally not enough to realize complete decellularization, the chemical method is more an enzymatic treatment method such as certain trypsin, and the enzymatic method may degrade some proteins in the decellularization process and has certain influence on the components of the method. The methods can change the biochemical composition of the tissue organ matrix under the influence of different degrees, and have the advantages of complex operation, higher cost, lower acellular degree and easy cell residue.
Based on the above, the invention provides a method for preparing acellular matrix hydrogel from biological material sources such as tissues, which is suitable for various tissues, has high acellular degree, can effectively reduce cell residues, and has small loss of tissue structures of raw materials.
Disclosure of Invention
In view of the above, there is a need to provide an acellular fluid, an acellular matrix hydrogel, and a preparation method and applications thereof.
In order to achieve the technical purpose, the technical scheme adopted by the application is as follows:
in a first aspect, the present invention provides a cell removal solution comprising the following components in final concentrations: NaCl or NH 4 Cl, 1-5 mol/L; 1-10% of sodium deoxycholate; DMSO, 0.5-5%; 0.1 to 5 percent of tributyl phosphate; 0.01-0.5mol/L of glycine; the solvent is water.
Preferably, the cell removal solution further comprises: 0.1-2% of Triton X or Tween-20 or NP-40.
Further, the cell removal liquid also comprises a pH regulator.
Further, the pH regulator is hydrochloric acid, and the pH of the cell removal liquid is regulated to 2-5.
In a second aspect, the present invention provides a method for preparing an acellular matrix hydrogel, comprising the steps of:
step 1, preprocessing collected tissue blocks;
step 2, shearing the tissue blocks obtained in the step 1, and then placing the sheared tissue blocks in the cell removing liquid to be stirred to obtain a cell removing scaffold;
step 3, cleaning the acellular scaffold, freeze-drying, and grinding into matrix powder after freeze-drying;
step 4, digesting the matrix powder by using digestive juice to obtain pre-gel;
and 5, filtering out undigested impurities in the pre-gel, and then adjusting the pH value to 6.5-7.5 to obtain the acellular matrix hydrogel.
Further, the pretreatment process in step 1 includes impurity removal, cleaning, and surface disinfection.
Further, in the step 2, the tissue blocks are stirred in the cell removal liquid at normal temperature for 4-8 h.
Further, the acellular scaffold in the step 3 is frozen for more than 2 hours in an environment of minus 80 +/-5 ℃ before being frozen.
Preferably, the acellular scaffold in the step 3 is frozen for 2-4 hours in an environment of-80 +/-5 ℃ before being freeze-dried.
Further, the lyophilization temperature is-80 ± 5 ℃.
Furthermore, the digestive juice is pepsin digestive juice containing hydrochloric acid, and the digestion time is 2-48 h.
Further, step 5 was performed by filtering undigested impurities from the pre-gel using a 22uM microfiltration membrane.
Further, in step 5, 0.5-5M sodium hydroxide solution is used to adjust the pH.
In a third aspect, the invention provides an acellular matrix hydrogel obtained by the above preparation method.
In a fourth aspect, the present invention provides the use of the acellular matrix hydrogel prepared by the above method in organoid culture.
The invention has the beneficial effects that:
1. the acellular fluid and the acellular matrix hydrogel disclosed by the invention are simple in components, simple in preparation process, good in repeatability, suitable for various tissues, high in acellular degree, short in preparation time, capable of furthest keeping the integrity of the internal structure of the acellular matrix, small in loss of the tissue structure of raw materials, capable of effectively reducing cell residues and providing a foundation for the application of the acellular matrix hydrogel.
2. The acellular matrix hydrogel disclosed by the invention is used for organoid culture, has good biocompatibility with organoid tissues and cells, has a good tissue repair effect, can be better compatible with the cells, and is favorable for survival and proliferation of the cells cultured by 3D.
Drawings
FIG. 1 is a pictorial representation of an acellular matrix hydrogel of example 1;
FIG. 2 is a gel-forming diagram of the acellular matrix hydrogel of example 2 dropped on a petri dish;
FIG. 3 is a photomicrograph of the mouse intestinal organoids cultured in example 4;
FIG. 4 is a photomontage of lung organoids of mice cultured in example 5;
FIG. 5 is a photomicrograph of the colon cancer organoids cultured in example 6.
Detailed Description
In the description of the present invention, the percentages are mass percentages.
In the description of the present invention, it is to be noted that those whose specific conditions are not specified in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturers. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The invention provides a cell removal liquid which comprises the following components in final concentration: NaCl or NH 4 Cl, 1-5 mol/L; 1-10% of sodium deoxycholate; DMSO, 0.5-5%; 0.1-5% of tributyl phosphate; 0.01-0.5mol/L of glycine; the solvent is water. Tributyl phosphate and glycine are added into the cell removal liquid to improve the cell removal efficiency.
In a preferred embodiment of the present invention, the cell removal solution further comprises: 0.1-2% of Triton X or Tween-20 or NP-40. One of the above components is further added into the cell removing liquid to form a system containing tributyl phosphate-glycine-Triton X/Tween-20/NP-40, which helps to further reduce the cell residue.
In an embodiment of the invention, the decellularization fluid further comprises a pH adjusting agent. Hydrochloric acid is used as a pH regulator to regulate the pH of the cell removal solution to 2-5, so that the cell removal in a system is facilitated under an acidic environment, and the cell removal efficiency is improved.
The invention also provides a preparation method of the acellular matrix hydrogel, which comprises the following steps:
step 1, preprocessing collected tissue blocks;
step 2, shearing the tissue block obtained in the step 1, and then placing the tissue block in the acellular liquid for stirring treatment to obtain an acellular scaffold;
step 3, cleaning the acellular scaffold, freeze-drying, and grinding into matrix powder after freeze-drying;
step 4, digesting the matrix powder by using digestive juice to obtain pre-gel;
and 5, filtering out undigested impurities in the pre-gel, and then adjusting the pH value to 6.5-7.5 to obtain the acellular matrix hydrogel.
In the embodiment of the present invention, the pretreatment process in step 1 includes processes of removing impurities, cleaning, surface sterilization, and the like.
In the embodiment of the invention, the tissue block in the step 2 is stirred in the cell removal liquid at normal temperature for 4-8h to ensure that the tissue block and the cell removal liquid are in full contact reaction to form the cell removal scaffold.
In the embodiment of the invention, the decellularized scaffold in the step 3 is frozen in an environment of minus 80 +/-5 ℃ for more than 2 hours before being frozen. Pre-freezing helps to preserve the mechanical properties of the decellularized scaffold.
In a preferred embodiment of the present invention, the decellularized scaffold in step 3 is frozen for 2-4h in an environment of-80. + -. 5 ℃ before being lyophilized.
In the examples of the present invention, the lyophilization temperature is-80 ± 5 ℃.
In the embodiment of the invention, the digestive juice is pepsin digestive juice containing hydrochloric acid, and the digestion time is 2-48 h.
In the present example, 22uM microfiltration was used to filter out undigested impurities from the pre-gel in step 5.
In the examples of the present invention, the pH is adjusted in step 5 with 0.5-5M sodium hydroxide solution.
The invention provides an acellular matrix hydrogel which is obtained by adopting the preparation method.
In addition, the invention provides application of the acellular matrix hydrogel prepared by the method in organoid culture.
The present invention will now be described in further detail with reference to the following figures and specific examples, which are intended to be illustrative, but not limiting, of the invention.
Example 1
This example provides a method for preparing a porcine small intestine mucosal acellular matrix hydrogel, comprising the following steps:
step 1, taking fresh pig small intestine tissue, cutting off fat tissue and partial outer membrane on the surface, washing in distilled water, and then carrying out surface disinfection;
step 2, shearing the porcine small intestine tissue block obtained in the step 1, and then placing the sheared porcine small intestine tissue block into a cell removal solution, wherein the cell removal solution comprises the following components in final concentration: 1mol/L NaCl, 1% sodium deoxycholate, 0.5% DMSO, 0.1% tributyl phosphate, 0.1mol/L glycine and 0.1% Triton X, stirring at 80r/min for 4 hours, and adjusting the pH of the cell-free solution to 3 with hydrochloric acid to obtain a cell-free scaffold;
step 3, washing the obtained acellular scaffold by using deionized water;
step 4, freezing the washed acellular scaffold in a refrigerator at minus 80 +/-5 ℃ for 2 hours, transferring the acellular scaffold into a freeze dryer at minus 80 +/-5 ℃ for freeze-drying, and shearing and grinding the acellular scaffold into powder to obtain matrix powder;
step 5, transferring the matrix powder into a solution containing hydrochloric acid pepsin, stirring and digesting for 4 hours at room temperature to obtain a pre-gel;
step 6, using a 22uM microporous filter membrane to carry out suction filtration to remove undigested impurities in the pre-gel;
and 7, adjusting the pH of the pre-gel to 7 by using sodium hydroxide to obtain the acellular matrix hydrogel, wherein the obtained acellular matrix hydrogel is shown in figure 1.
And 8, carrying out DNA concentration detection on the obtained acellular matrix hydrogel according to the instruction of the Qubit dsDNA HS Assay Kit, wherein the detection result is 0.02ng/ul, the residual DNA is very low, and the result indicates that the acellular treatment can effectively reduce the cell residue.
Example 2
The embodiment provides a preparation method of a pig placenta acellular matrix hydrogel, which comprises the following steps:
step 1, taking fresh pig placenta tissue, cutting off fat tissue and partial outer membrane on the surface, washing in distilled water, and then carrying out surface disinfection;
step 2, shearing the pig placenta tissue blocks obtained in the step 1, and then placing the cut pig placenta tissue blocks into a cell removal solution, wherein the cell removal solution comprises the following components in final concentration: 3mol/L NH 4 Stirring Cl, 6% of sodium deoxycholate, 3% of DMSO, 3% of tributyl phosphate, 0.2mol/L of glycine and 1% of Tween-20 at the mass concentration of 100r/min for 8 hours, and adjusting the pH value to 4 by using hydrochloric acid to obtain the acellular scaffold;
step 3, washing the obtained acellular scaffold by using deionized water;
step 4, freezing the washed acellular scaffold in a refrigerator at minus 80 +/-5 ℃ for 2 hours, transferring the acellular scaffold into a freeze dryer at minus 80 +/-5 ℃ for freeze-drying, and shearing and grinding the acellular scaffold into powder to obtain matrix powder;
step 5, transferring the matrix powder into a solution containing hydrochloric acid pepsin, stirring and digesting for 24 hours at room temperature to obtain a pre-gel;
step 6, using a 22uM microporous filter membrane to carry out suction filtration to remove undigested impurities in the pre-gel;
and 7, adjusting the pH of the pre-gel to 7 by using sodium hydroxide to obtain acellular matrix hydrogel, wherein the obtained acellular matrix hydrogel is dripped into a culture dish to be solidified, and the state is shown in figure 2.
And 8, detecting the DNA concentration of the obtained acellular matrix hydrogel according to the instruction of the Qubit dsDNA HS Assay Kit, wherein the detection result is that the DNA is not detected due to too low concentration, and the acellular treatment can be basically free of cell residues.
Example 3
The embodiment provides a preparation method of pig umbilical cord acellular matrix glue, which comprises the following steps:
step 1, taking fresh pig umbilical cord tissue, cutting off fat tissue and partial outer membrane on the surface, washing in distilled water, and then carrying out surface disinfection;
step 2, shearing the pig umbilical cord tissue block obtained in the step 1, and then placing the cut pig umbilical cord tissue block into a cell removal liquid, wherein the cell removal liquid comprises the following components in final concentration: 5mol/L NaCl, 8% sodium deoxycholate by mass concentration, 5% DMSO, 5% tributyl phosphate, 0.5mol/L glycine and 2% NP-40, stirring at 50r/min for 6h, and adjusting the pH value to 4 with hydrochloric acid to obtain a decellularized scaffold;
step 3, washing the obtained acellular scaffold by using deionized water;
step 4, freezing the washed acellular scaffold in a refrigerator at minus 80 +/-5 ℃ for 2 hours, transferring the acellular scaffold into a freeze dryer at minus 80 +/-5 ℃ for freeze-drying, and shearing and grinding the acellular scaffold into powder to obtain matrix powder;
step 5, transferring the matrix powder into a solution containing hydrochloric acid pepsin, stirring and digesting for 8 hours at room temperature to obtain a pre-gel;
step 6, using a 22uM microporous filter membrane to carry out suction filtration to remove undigested impurities in the pre-gel;
and 7, adjusting the pH of the pre-gel to 7 by using sodium hydroxide to obtain the acellular matrix hydrogel, and thus obtaining the acellular matrix hydrogel.
And 8, detecting the DNA concentration of the obtained acellular matrix hydrogel according to the instruction of the Qubit dsDNA HS Assay Kit, wherein the DNA concentration of the detection result is 0.01ng/ul, and the residual DNA is very low, which indicates that the acellular treatment can be basically carried out without cell residues.
Example 4
Application of small intestine mucosa acellular matrix gum in intestinal organoid culture
This example provides a method for organoid culture using a small intestine mucosal acellular matrix hydrogel, using the porcine small intestine mucosal acellular matrix hydrogel of example 1, comprising the steps of:
1) the fresh mouse intestine is pretreated, washed several times with 1% double antibody-containing physiological saline, freed from blood clots and other impurities, and cut up thoroughly.
2) Blowing off the tissue block, filtering to obtain cell mass with diameter of 15-100 um, centrifuging to remove supernatant, and counting cells to 2 × 10 5 Individual cells are used for organoid culture.
3) An appropriate amount of mouse intestinal organoid medium (Chuangguo International Biotechnology (Guangzhou) Co., Ltd., product No. M501) was taken to resuspend the cells, and an appropriate amount of the matrix hydrogel prepared in example 1 was mixed with the cell suspension on ice with a precooled pipette tip to prepare a cell-gel mixed solution.
4) The cell-gel mixture was dropped into a 60mm petri dish using a pipette to form 30-50 ul/drop gel drops.
5) Putting the culture dish after the glue dripping into CO 2 Standing in the incubator for 2min, carefully reversing after no obvious flow of light shaking glue drops, and fully solidifying for 30 min.
6) 3ml of the matrix hydrogel of example 1 was added to the petri dish, which was then placed in a thermostatted incubator at 37 ℃ with 5% CO 2 Culturing at the concentration.
7) Replacing the culture medium every 2 days, culturing for 6 days to obtain mouse intestinal organoid with average diameter of 80-100 μm, and observing tissue morphology structure under a common optical microscope as shown in figure 3. After counting the cells, the total number of cells was 1.5X 10 6 The number of the cells was 7.5 times larger than that before the culture.
The hydrogels of example 2 and example 3 were used in this example to obtain the same culturing effect.
Example 5
Application of placenta acellular matrix glue to lung organoid culture
This example provides a method for culturing organoids using the acellular matrix hydrogel of placental mucosa using the acellular matrix hydrogel of porcine placental mucosa of example 2, comprising the steps of:
1) the method comprises pretreating fresh mouse lung, washing with 1% double antibody-containing physiological saline for several times, removing blood clot and other impurities, and sufficiently mincing.
8) Digesting, blowing off and filtering the tissue block to obtain cell mass with diameter of 15-100 um, centrifuging to remove supernatant, and counting cells to obtain 2 × 10 5 Individual cells are used for organoid culture.
2) An appropriate amount of mouse lung organoid medium (Chuanghou International Biotechnology, Inc., product number M504) was taken to resuspend the cells, and an appropriate amount of the matrix hydrogel prepared in example 2 was mixed with the cell suspension on ice using a pre-cooled pipette tip to prepare a cell-gel mixture.
3) The cell-gel mixture was dropped into a 60mm petri dish using a pipette to form 30-50 ul/drop gel drops.
4) Putting the culture dish after the glue dripping into CO 2 Standing in the incubator for 2min, carefully reversing after no obvious flow of light shaking glue drops, and fully solidifying for 30 min.
5) 3ml of the matrix hydrogel of example 2 was added to the petri dish, which was then placed in a thermostatted incubator at 37 ℃ with 5% CO 2 Culturing under the concentration.
6) The culture medium was changed every 2 days, and after 7 days of culture, mouse lung organoids with mean diameter over 100 μm were obtained, and the morphological structure of the tissue observed under a normal optical microscope is shown in FIG. 4. After counting the cells, the total number of cells was 3.4X 10 6 The number of the cells was increased 17-fold compared with that before the culture.
The hydrogels of example 1 and example 3 were used in this example to obtain the same culturing effect.
Example 6
Application of umbilical cord acellular matrix glue to culture of intestinal cancer organoid
This example provides a method for organoid culture using umbilical cord mucosal acellular matrix hydrogel, using the umbilical cord mucosal acellular matrix hydrogel of example 3, comprising the steps of:
1) pretreating fresh intestinal cancer tissue, washing with 1% double antibody-containing physiological saline for several times, removing blood clot and other impurities, and sufficiently cutting.
2) Digesting, blowing off and filtering the tissue block to obtain cell mass with diameter of 15-100 um, centrifuging to remove supernatant, and counting cells to obtain 2 × 10 5 Individual cells are used for organoid culture.
3) An appropriate amount of intestinal cancer organoid medium (Chuangguo International Biotechnology, Inc., Guangzhou, Cat. No. M102) was taken to resuspend the cells, and an appropriate amount of the matrix hydrogel prepared in example 3 was mixed with the cell suspension on ice using a pre-cooled pipette tip to prepare a cell-gel mixture.
4) The cell-gel mixture was dropped into a 60mm petri dish using a pipette to form 30-50 ul/drop gel drops.
5) Putting the culture dish after the glue dripping into CO 2 Standing in the incubator for 2min, carefully reversing after no obvious flow of light shaking glue drops, and fully solidifying for 30 min.
6) 3ml of the matrix hydrogel of example 3 was added to the petri dish, which was then placed in a thermostatted incubator at 37 ℃ with 5% CO 2 Culturing at the concentration.
7) Replacing the culture medium every 2 days, culturing for 6 days to obtain intestinal cancer organoids with average diameter of 50-100 μm, and observing the tissue morphology structure under a common optical microscope as shown in figure 5. After counting the cells, the total number of cells was 8.4X 10 6 The number of the cells was increased by 42 times as much as that before the culture.
The hydrogels of example 1 and example 2 were used in this example to obtain the same culturing effect.
Comparative example 1
0.1mol/L glycine in the cell-free solution of example 1 was removed, and the same conditions as in example 1 were used. The DNA concentration in the acellular matrix hydrogel is measured according to the method of example 1, the DNA concentration is 7.58ng/ul, and the residual DNA is far higher than that in example 1, which shows that the addition of glycine is favorable for improving the acellular efficiency.
Comparative example 2
0.1mol/L of tributyl phosphate in the cell-free solution of example 1 was removed, and the same conditions as in example 1 were used. The DNA concentration in the acellular matrix hydrogel is measured according to the method in example 1, the DNA concentration is 5.4ng/ul, and the residual DNA is far higher than that in example 1, which shows that the addition of tributyl phosphate is favorable for improving the acellular efficiency.
Comparative example 3
The pH of the cell-free solution in example 1 was adjusted to 7.5, and the same conditions as in example 1 were used. When the DNA concentration in the acellular matrix hydrogel is measured according to the method in example 1, the DNA concentration is 2.96ng/ul, the residual DNA is far higher than that in example 1, and the condition that the pH is acidic is proved to be favorable for improving the acellular efficiency.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (10)

1. A cell removal fluid, comprising: comprising the following components in final concentrations: NaCl or NH 4 Cl, 1-5 mol/L; 1-10% of sodium deoxycholate; DMSO, 0.5-5%; 0.1 to 5 percent of tributyl phosphate; 0.01-0.5mol/L of glycine; the solvent is water.
2. A cell removal fluid as claimed in claim 1, wherein: the cell removal solution further comprises: 0.1-2% of Triton X or Tween-20 or NP-40.
3. A cell removal fluid according to claim 1 or 2, wherein: the cell removal solution also comprises a pH regulator.
4. A cell removal fluid as claimed in claim 3, wherein: the pH regulator is hydrochloric acid, and regulates the pH of the cell-free liquid to 2-5.
5. A preparation method of acellular matrix hydrogel is characterized by comprising the following steps: the method comprises the following steps:
step 1, preprocessing collected tissue blocks;
step 2, shearing the tissue blocks obtained in the step 1, and then placing the sheared tissue blocks in the cell removing liquid to be stirred to obtain a cell removing scaffold;
step 3, cleaning the acellular scaffold, freeze-drying, and grinding into matrix powder after freeze-drying;
step 4, digesting the matrix powder by using digestive juice to obtain pre-gel;
and 5, filtering out undigested impurities in the pre-gel, and then adjusting the pH value to 6.5-7.5 to obtain the acellular matrix hydrogel.
6. The method of claim 5, wherein the method comprises the steps of: in the step 2, the tissue blocks are stirred in the cell removal liquid at normal temperature for 4-8 h.
7. The method of claim 5, wherein the method comprises the steps of: freezing the acellular scaffold in the step 3 for more than 2 hours in an environment of minus 80 +/-5 ℃ before freeze-drying.
8. The method of claim 7, wherein the method comprises the steps of: freezing the acellular scaffold in the step 3 for 2-4 hours in an environment of minus 80 +/-5 ℃ before freeze-drying.
9. An acellular matrix hydrogel, comprising: is obtained by the preparation method of any one of claims 5 to 8.
10. Use of the acellular fluid according to any one of claims 1 to 4, the acellular matrix hydrogel obtained by the preparation method according to any one of claims 5 to 8, or the acellular matrix hydrogel according to claim 9 in organoid culture.
CN202210458274.XA 2022-04-27 2022-04-27 Acellular fluid, acellular matrix hydrogel, and preparation methods and application thereof Active CN114908034B (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130028981A1 (en) * 2010-02-26 2013-01-31 Decell Technologies Inc. Methods for tissue decellularization
CN112089890A (en) * 2020-08-28 2020-12-18 广东乾晖生物科技有限公司 Acellular matrix hydrogel and preparation method and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130028981A1 (en) * 2010-02-26 2013-01-31 Decell Technologies Inc. Methods for tissue decellularization
CN112089890A (en) * 2020-08-28 2020-12-18 广东乾晖生物科技有限公司 Acellular matrix hydrogel and preparation method and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KISHOR TARDALKAR等: "Novel Approach Toward the Generation of Tissue Engineered Heart Valve by Using Combination of Antioxidant and Detergent:A Potential Therapy in Cardiovascular Tissue Engineering", 《TISSUE ENG REGEN MED 》 *

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