阅读说明：本技术 一种水凝胶、制备方法及其三维细胞培养方法 (Hydrogel, preparation method and three-dimensional cell culture method thereof ) 是由 孙晓娇 张文平 张红霞 刘星 陆重益 于 2020-04-12 设计创作，主要内容包括：本发明揭示了一种水凝胶、制备方法及其三维细胞培养方法,水凝胶的制备方法包括：步骤S1、制备猪脱细胞真皮和/或猪脱细胞膀胱；步骤S2、冻干所述猪脱细胞真皮和/或所述猪脱细胞膀胱；步骤S3、研磨冻干的所述猪脱细胞真皮和/或冻干所述猪脱细胞膀胱成细颗粒；步骤S4、将所述细颗粒加入胃蛋白酶消化液中,搅拌48h-72h,形成细胞外基质支架；以及步骤S5、调节所述胃蛋白酶消化液的PH值和盐浓度,使得所述胃蛋白酶灭活,所述细胞外基质支架中的蛋白质进行重组形成所述水凝胶。(The invention discloses a hydrogel, a preparation method and a three-dimensional cell culture method thereof, wherein the preparation method of the hydrogel comprises the following steps: step S1, preparing pig acellular dermis and/or pig acellular bladder; step S2, lyophilizing the porcine acellular dermis and/or the porcine acellular bladder; step S3, grinding the lyophilized porcine acellular dermis and/or lyophilizing the porcine acellular bladder into fine particles; step S4, adding the fine particles into the pepsin digestive juice, and stirring for 48-72h to form an extracellular matrix support; and S5, adjusting the pH value and the salt concentration of the pepsin digestive juice to inactivate the pepsin, and recombining the proteins in the extracellular matrix scaffold to form the hydrogel.)

1. A method for preparing a hydrogel for three-dimensional cell culture, comprising:

step S1, preparing pig acellular dermis and/or pig acellular bladder;

step S2, lyophilizing the porcine acellular dermis and/or the porcine acellular bladder;

step S3, grinding the lyophilized porcine acellular dermis and/or lyophilizing the porcine acellular bladder into fine particles;

step S4, adding the fine particles into the pepsin digestive juice, and stirring for 48-72h to form an extracellular matrix support; and

and step S5, adjusting the pH value and the salt concentration of the pepsin digestive juice to inactivate the pepsin, and recombining the proteins in the extracellular matrix scaffold to form the hydrogel.

2. The method for preparing hydrogel for three-dimensional cell culture according to claim 1, wherein the step S1 further comprises: step S11: a porcine acellular dermal raw material treatment comprising:

collecting full thickness skin slices from market-weight adult pigs; and

the full thickness skin sections were cut into rectangular sections of 35cm × 50cm, and the rectangular sections of skin were separated from the porcine dermis layer for subcutaneous fat and connective tissue layers.

3. The method for preparing hydrogel for three-dimensional cell culture according to claim 2, wherein the porcine dermal layer is stored in a freezing chamber at-80 ℃ before decellularization and is removed and thawed to room temperature before decellularization.

4. The method of preparing a hydrogel for three-dimensional cell culture according to claim 2, wherein the full-thickness skin section is collected from the dorsal lateral side of an adult pig.

5. The method for preparing hydrogel for three-dimensional cell culture according to claim 2, wherein the step S1 further comprises: step S12: the method for preparing the porcine acellular dermis by carrying out acellular treatment on the porcine dermis comprises the following steps:

step S121: placing the porcine dermis layer in a conical flask containing 0.25% trypsin solution, and stirring the porcine dermis layer for 6 hours on an orbital shaker at 300 rpm;

step S122: removing 0.25% trypsin solution, sequentially placing the corium layer of the pig subjected to enzymolysis treatment in a conical flask containing redistilled water, and washing for three times on an orbital shaker at 300 revolutions per minute, wherein the washing time is 15 minutes each time; stirring in an erlenmeyer flask containing 70% ethanol solution for 10 hours on an orbital shaker at 300 rpm; stirring in an erlenmeyer flask containing 3% hydrogen peroxide solution for 15 minutes on an orbital shaker at 300 rpm; soaking twice in conical flask containing redistilled water on 300 r/min orbital shaker for 15 min each time;

step S123: placing the porcine dermis treated in the step 122 into an erlenmeyer flask containing Triton X-100 solution, and stirring for 16 hours on an orbital shaker at 300 revolutions per minute; removing the used Triton X-100 solution from the Erlenmeyer flask, injecting fresh Triton X-100 solution, and stirring for 16 hr on 300 rpm orbital shaker;

step S124: placing the porcine dermis layer treated in the step S123 into a conical flask containing redistilled water, and washing the porcine dermis layer for three times on an orbital shaker at 300 revolutions per minute, wherein each time is 15 minutes;

step S125: placing the porcine dermal layer treated in the step S124 into a conical flask containing a peroxyacetic acid solution, and shaking the conical flask on an orbital shaker at the speed of 300 revolutions per minute for 2 hours to prepare porcine acellular dermis;

step S126: placing the pig acellular dermis in a conical flask containing 1X phosphate buffer salt solution in sequence, and washing twice on an orbital shaker at 300 revolutions per minute for 15 minutes each time; washing the decellularized pig dermis in an erlenmeyer flask containing redistilled water on an orbital shaker at 300 rpm for 15 minutes;

step S127: the pig decellularized dermis treated in step S126 is stored in a freezer at-80 ℃, and is taken out and thawed to room temperature before use.

6. The method of claim 5, wherein the volume of the 0.25% trypsin solution is 20 times the weight of the dermis layer of the pig.

7. The method of claim 5, wherein the Triton X-100 solution is a mixture of 1% Triton X-100 and 0.26% EDTA and 0.69% Tris buffered saline.

8. The method for preparing hydrogel for three-dimensional cell culture according to claim 1, wherein the step S1 further comprises: step S13: a porcine decellularized bladder raw material treatment comprising:

collecting adult pig bladder from market;

cutting off the top end and the neck of the pig bladder; then cutting into a rectangular cut along the midline of the connective tissue, wherein the rectangular cut can be laid flat with the lumen side facing downwards; stretching the rectangular cut blocks to increase the area to two times of the original area by using a hard plastic scraper; placing the cut rectangular blocks on the surfaces of the stretched rectangular blocks by using scissors, and cutting along the central line of the pig bladder; stripping off muscle and mucosa, and keeping basement membrane and primary epithelial membrane of pig bladder.

9. The method for preparing hydrogel for three-dimensional cell culture according to claim 8, wherein the thickness of basement membrane and primary epithelial membrane of porcine bladder is 100-150 μm.

10. The method for preparing hydrogel for three-dimensional cell culture according to claim 8, wherein the step S1 further comprises: step S14: the pig acellular bladder is prepared by carrying out acellular treatment on a pig bladder basement membrane and a protoderm membrane, wherein the acellular treatment on the pig bladder basement membrane and the protoderm membrane comprises the following steps:

step S141: placing the porcine bladder basement membrane and the original membrane in a conical flask containing peroxyacetic acid solution, and placing the conical flask on a 300 r/m orbital shaker for 2 hours to prepare the porcine acellular bladder;

step S142: washing the decellularized bladder of the pig twice by using 1X phosphate buffer saline solution on an orbital shaker at 300 r/m for 15 minutes each time; washing the decellularized bladder of the pig twice by using redistilled water, wherein each time lasts for 15 minutes; and

step S143: the porcine decellularized bladder was stored in a freezer at-80 ℃.

11. The method for preparing the hydrogel for three-dimensional cell culture according to claim 10, wherein the volume of the peroxyacetic acid solution used is 20 times the weight of the porcine bladder basement membrane and the protomembrane.

12. The method for preparing the hydrogel for three-dimensional cell culture according to claim 5 or 10, wherein the peracetic acid solution is a 0.1% peracetic acid solution, and the 0.1% peracetic acid solution contains 4% ethanol.

13. The method for producing a hydrogel for three-dimensional cell culture according to claim 1,

the step S2 comprises taking out the frozen pig acellular dermis and/or pig acellular bladder from a-80 ℃ refrigerator, and immediately putting the pig acellular dermis and/or pig acellular bladder into a freeze dryer for freeze-drying; and

the step S3 includes grinding the freeze-dried porcine acellular dermis and/or porcine acellular bladder into fine particles with a grinder of 40 mesh screen.

14. The method for preparing a hydrogel for three-dimensional cell culture according to claim 13,

the step S4 includes magnetically stirring the fine particles and pepsin digestion solution with a concentration of 1mg/m L at room temperature for 48-72 hours until the fine particles are completely digested to form extracellular matrix scaffolds.

15. The method for producing a hydrogel for three-dimensional cell culture according to claim 14,

the step S5 includes: adjusting the pH value of the pepsin digestion solution by adding NaOH solution with the concentration of 0.1N and hydrochloric acid solution with the concentration of 0.1N in the pepsin digestion solution, and adjusting the salt concentration by adding 10X phosphate buffer salt solution or 1X phosphate buffer salt solution so that the proteins in the extracellular matrix scaffold are recombined to form the hydrogel.

16. A hydrogel suitable for three-dimensional cell culture, wherein the hydrogel is prepared by using the method for preparing a hydrogel for three-dimensional cell culture according to any one of claims 1 to 15.

17. A three-dimensional cell culture method for hydrogel surfaces, which is characterized by comprising the following steps:

step S10: preparing a hydrogel as defined in claim 16 under sterile conditions;

step S20, implanting NIH 3T3 fibroblast cell line or C2C12 myoblast cell line onto the hydrogel, wherein the seeding density of the cells on the hydrogel is 5.0 × 105 cells/cm²；

Step S30: standing for 16 hours until NIH 3T3 fibroblasts or C2C12 myoblasts are fully attached to the surface of the hydrogel, and adding a culture medium flush with the hydrogel; and

step S40: the medium was changed every 3 days to supplement the nutrients required by NIH 3T3 fibroblasts or C2C12 myoblasts.

18. The method for three-dimensional cell culture on the surface of the hydrogel according to claim 17, wherein the step S20 further comprises a pretreatment of the hydrogel, the pretreatment comprising: placing the sterilized stainless steel ring into a 6-well plate, sucking 0.5 ml of newly prepared hydrogel solution into a 6-well plate culture dish by using a suction pipe, and covering the sterilized stainless steel ring; placing a 6-hole plate culture dish in a non-wetting incubator at 37 ℃ for 1h to solidify the hydrogel; the cured hydrogel was removed for use.

19. The three-dimensional cell culture method according to claim 17, wherein the step S20 further comprises preparing hydrogel with a concentration of 6mg/m L and hydrogel with a concentration of 8mg/m L.

20. A method of three-dimensional cell culture within a hydrogel, the method comprising:

step S100: preparing a single cell suspension;

step S200: preparing a hydrogel as claimed in claim 16 and formulating the hydrogel into a pre-gel solution;

step S300: uniformly mixing the single cell suspension and the pre-gel solution to form a mixture, and transferring the mixture into a culture dish containing a stainless steel ring;

step S400: placing the petri dish containing the mixture and stainless steel ring in a dry-heat incubator at 37 ℃ for 45 minutes, allowing the hydrogel to solidify, removing the stainless steel ring surrounding the solidified hydrogel, and adding sufficient fresh medium to cover the solidified hydrogel; and

step S500: the medium was changed every 3 days to supplement the nutrients required for cell viability.

21. The method for three-dimensional cell culture within a hydrogel according to claim 19,

the step S100: preparing a single cell suspension comprising:

taking out the cell culture bottle from the incubator after the cells are separated, and sucking the culture medium under the condition of not interfering the cells;

adding 2-3 ml of heated trypsin-EDTA mixed solution into the flask, and putting the flask into an incubator for 5-15 minutes;

after separating the cells, adding 6-8 ml of fresh cell culture medium into the flask to form a cell suspension;

transfer the cell suspension to a centrifuge tube and take an aliquot for counting and adjust the cell suspension to a final concentration of 1.0 × 10⁶Cells/m L hydrogel.

22. The method of claim 20, wherein the cells in the cell culture flask are NIH 3T3 fibroblasts or C2C12 myoblasts.

Technical Field

The invention relates to the technical field of biological materials, in particular to a hydrogel prepared based on decellularization, a preparation method thereof and a three-dimensional cell culture method using the hydrogel.

Background

It has long been thought that extracellular matrix (ECM) not only provides structural support, but also regulates cell growth, survival, maturation, differentiation and development of resident cells. Although many components of the ECM can be stored in different tissue types, each tissue is considered to have a unique composition.

Recently, ECM scaffolds derived from the ECM of various tissues (including skin, fat, pericardium, heart, skeletal muscle and liver) were observed to contribute to constructive remodeling or formation of tissues in situ when used as biomaterials in vivo and in vitro experiments. In some cases, these ECM scaffolds have a tissue-specific effect on cell behavior.

Despite the unique interactions between ECM and resident cells, two-dimensional in vitro cell studies typically assess cellular behavior on coatings consisting of either a single purified protein or directly on polystyrene culture dishes. These methods do not accurately mimic the complexity of the extracellular microenvironment and may significantly alter the results observed in vitro, making it difficult for in vitro studies to perform appropriate transformations in vivo.

The use of ECM matrices is becoming increasingly common in three-dimensional in vitro cell experiments, as these matrices are believed to better mimic the cellular natural environment and achieve appropriate cellular functions.

Disclosure of Invention

The invention aims to provide a method for preparing hydrogel by mechanical layered decellularization and a culture method using the hydrogel as a scaffold for subsequent three-dimensional culture of two cell types (NIH 3T3 fibroblasts and C2C12 myoblasts).

The present invention provides a method for preparing a hydrogel for three-dimensional cell culture, the method comprising:

step S1, preparing pig acellular dermis and/or pig acellular bladder;

step S2, lyophilizing the porcine acellular dermis and/or the porcine acellular bladder;

step S3, grinding the lyophilized porcine acellular dermis and/or lyophilizing the porcine acellular bladder into fine particles;

step S4, adding the fine particles into the pepsin digestive juice, and stirring for 48-72h to form an extracellular matrix support; and

and step S5, adjusting the pH value and the salt concentration of the pepsin digestive juice to inactivate the pepsin, and recombining the proteins in the extracellular matrix scaffold to form the hydrogel.

As an alternative technical scheme, the step S1 further comprises the step S11 of processing the pig acellular dermal raw material, wherein the processing comprises collecting full-thickness skin slices from commercial-weight adult pigs, cutting the full-thickness skin slices into rectangular blocks of 35cm × 50cm, and separating the subcutaneous fat and connective tissue layers of the skin of the rectangular blocks from the dermis layer of the pigs.

As an alternative, the porcine dermis layer may be stored in a freezer at-80 ℃ prior to decellularization and removed and thawed to room temperature prior to decellularization.

As an alternative solution, full thickness skin slices were collected from the dorsal lateral side of adult pigs.

As an optional technical solution, the step S1 further includes: step S12: the method for preparing the porcine acellular dermis by carrying out acellular treatment on the porcine dermis comprises the following steps: step S121: placing the porcine dermis layer in a conical flask containing 0.25% trypsin solution, and stirring the porcine dermis layer for 6 hours on an orbital shaker at 300 rpm; step S122: removing 0.25% trypsin solution, sequentially placing the corium layer of the pig subjected to enzymolysis treatment in a conical flask containing redistilled water, and washing for three times on an orbital shaker at 300 revolutions per minute, wherein the washing time is 15 minutes each time; stirring in an erlenmeyer flask containing 70% ethanol solution for 10 hours on an orbital shaker at 300 rpm; stirring in an erlenmeyer flask containing 3% hydrogen peroxide solution for 15 minutes on an orbital shaker at 300 rpm; soaking twice in conical flask containing redistilled water on 300 r/min orbital shaker for 15 min each time; step S123: placing the porcine dermis treated in the step 122 into an erlenmeyer flask containing Triton X-100 solution, and stirring for 16 hours on an orbital shaker at 300 revolutions per minute; removing the used Triton X-100 solution from the Erlenmeyer flask, injecting fresh Triton X-100 solution, and stirring for 16 hr on 300 rpm orbital shaker; step S124: placing the porcine dermis layer treated in the step S123 into a conical flask containing redistilled water, and washing the porcine dermis layer for three times on an orbital shaker at 300 revolutions per minute, wherein each time is 15 minutes; step S125: placing the porcine dermal layer treated in the step S124 into a conical flask containing a peroxyacetic acid solution, and shaking the conical flask on an orbital shaker at the speed of 300 revolutions per minute for 2 hours to prepare porcine acellular dermis; step S126: placing the pig acellular dermis in a conical flask containing 1X phosphate buffer salt solution in sequence, and washing twice on an orbital shaker at 300 revolutions per minute for 15 minutes each time; washing the decellularized pig dermis in an erlenmeyer flask containing redistilled water on an orbital shaker at 300 rpm for 15 minutes; step S127: the pig decellularized dermis treated in step S126 is stored in a freezer at-80 ℃, and is taken out and thawed to room temperature before use.

As an alternative, the volume of the 0.25% trypsin solution used is 20 times the weight of the porcine dermal layers.

As an optional technical scheme, the Triton X-100 solution is formed by mixing 1% Triton X-100 concentration solution, 0.26% EDTA concentration solution and 0.69% Tris buffer salt solution.

As an optional technical solution, the step S1 further includes: step S13: a porcine decellularized bladder raw material treatment comprising: collecting adult pig bladder from market; cutting off the top end and the neck of the pig bladder; then cutting into a rectangular cut along the midline of the connective tissue, wherein the rectangular cut can be laid flat with the lumen side facing downwards; stretching the rectangular cut blocks to increase the area to two times of the original area by using a hard plastic scraper; placing the cut rectangular blocks on the surfaces of the stretched rectangular blocks by using scissors, and cutting along the central line of the pig bladder; stripping off muscle and mucosa, and keeping basement membrane and primary epithelial membrane of pig bladder.

As an optional technical scheme, the thickness of the basement membrane and the primary epithelial membrane of the porcine bladder is 100-150 μm.

As an optional technical solution, the step S1 further includes: step S14: the pig acellular bladder is prepared by carrying out acellular treatment on a pig bladder basement membrane and a protoderm membrane, wherein the acellular treatment on the pig bladder basement membrane and the protoderm membrane comprises the following steps: step S141: placing the porcine bladder basement membrane and the original membrane in a conical flask containing peroxyacetic acid solution, and placing the conical flask on a 300 r/m orbital shaker for 2 hours to prepare the porcine acellular bladder; step S142: washing the decellularized bladder of the pig twice by using 1X phosphate buffer saline solution on an orbital shaker at 300 r/m for 15 minutes each time; washing the decellularized bladder of the pig twice by using redistilled water, wherein each time lasts for 15 minutes; and step S143: the porcine decellularized bladder was stored in a freezer at-80 ℃.

As an alternative technical scheme, the volume of the peroxyacetic acid solution is 20 times of the weight of the porcine bladder basement membrane and the protomembrane.

As an optional technical scheme, the peracetic acid solution is 0.1% in concentration, and the 0.1% in concentration contains 4% ethanol.

As an alternative technical solution, the step S2 includes taking the frozen pig acellular dermis and/or pig acellular bladder out of a refrigerator at-80 ℃, and immediately putting the pig acellular dermis and/or pig acellular bladder into a freeze dryer for freeze-drying; and the step S3 includes grinding the freeze-dried porcine acellular dermis and/or porcine acellular bladder into fine particles with a grinder of 40 mesh screen.

As an alternative technical scheme, the step S4 includes that the fine particles and pepsin digestive liquid with the concentration of 1mg/m L are magnetically stirred for 48-72 hours under the condition of room temperature until the fine particles are completely digested to form an extracellular matrix scaffold.

As an optional technical solution, the step S5 includes: adjusting the pH value of the pepsin digestion solution by adding NaOH solution with the concentration of 0.1N and hydrochloric acid solution with the concentration of 0.1N in the pepsin digestion solution, and adjusting the salt concentration by adding 10X phosphate buffer salt solution or 1X phosphate buffer salt solution so that the proteins in the extracellular matrix scaffold are recombined to form the hydrogel.

The present invention also provides a hydrogel suitable for three-dimensional cell culture, which is prepared using the method for preparing a hydrogel for three-dimensional cell culture as described above.

The present invention still further provides a three-dimensional cell culture method for a hydrogel surface, the three-dimensional cell culture method comprising:

step S10: preparing a hydrogel as defined in claim 16 under sterile conditions;

step S20, implanting NIH 3T3 fibroblast cell line or C2C12 myoblast cell line onto the hydrogel, wherein the seeding density of the cells on the hydrogel is 5.0 × 105 cells/cm²；

Step S30: standing for 16 hours until NIH 3T3 fibroblasts or C2C12 myoblasts are fully attached to the surface of the hydrogel, and adding a culture medium flush with the hydrogel; and

step S40: the medium was changed every 3 days to supplement the nutrients required by NIH 3T3 fibroblasts or C2C12 myoblasts.

As an optional technical solution, the step S20 further includes a hydrogel pretreatment, where the hydrogel pretreatment includes: placing the sterilized stainless steel ring into a 6-well plate, sucking 0.5 ml of newly prepared hydrogel solution into a 6-well plate culture dish by using a suction pipe, and covering the sterilized stainless steel ring; placing a 6-hole plate culture dish in a non-wetting incubator at 37 ℃ for 1h to solidify the hydrogel; the cured hydrogel was removed for use.

As an optional technical scheme, the step S20 further comprises the steps of preparing hydrogel with the concentration of 6mg/m L and hydrogel with the concentration of 8mg/m L.

As an alternative technical solution, the three-dimensional cell culture method includes:

step S100: preparing a single cell suspension;

step S200: preparing a hydrogel as claimed in claim 16 and formulating the hydrogel into a pre-gel solution;

step S300: uniformly mixing the single cell suspension and the pre-gel solution to form a mixture, and transferring the mixture into a culture dish containing a stainless steel ring;

step S400: placing the petri dish containing the mixture and stainless steel ring in a dry-heat incubator at 37 ℃ for 45 minutes, allowing the hydrogel to solidify, removing the stainless steel ring surrounding the solidified hydrogel, and adding sufficient fresh medium to cover the solidified hydrogel; and

step S500: the medium was changed every 3 days to supplement the nutrients required for cell viability.

As an alternative technical scheme, the step S100 of preparing the single cell suspension comprises the steps of taking out a cell culture bottle from an incubator to be separated, sucking a culture medium under the condition that the cells are not interfered, adding 2-3 ml of heated trypsin-EDTA mixed solution into the flask, putting the flask into the incubator for 5-15 minutes, adding 6-8 ml of fresh cell culture medium into the flask after the cells are separated to form the cell suspension, transferring the cell suspension into a centrifuge tube, taking an aliquot for counting, and adjusting the cell suspension to the final concentration of 1.0 × 10⁶Cells/m L hydrogel.

As an alternative technical scheme, the cells in the cell culture bottle are NIH 3T3 fibroblasts or C2C12 myoblasts.

The invention provides a method for preparing hydrogel after mechanical layering and decellularization of pig dermis and pig bladder, and the prepared hydrogel can be used for subsequent three-dimensional (3D) culture of two cell types (NIH 3T3 fibroblasts and C2C12 myoblasts). Although the hydrogel preparation methods described are designed for specific tissues and cell types, the basic principles can be applied to the decellularization and hydrogel preparation of a variety of other tissue types, as well as the subsequent formation of 3D cell culture models.

Drawings

FIG. 1 is a photograph showing the process of forming hydrogel in decellularized bladder of swine according to the present invention.

FIG. 2 is a photograph of the hydrogel of the present invention as cured.

Detailed Description

The present invention will be described in detail below with reference to specific embodiments shown in the drawings. These embodiments are not intended to limit the present invention, and structural, methodological, or functional changes made by those skilled in the art according to these embodiments are included in the scope of the present invention.

An object of the present invention is to provide a method for preparing a hydrogel for three-dimensional cell culture, the method comprising:

step S1, preparing pig acellular dermis and/or pig acellular bladder;

step S2, lyophilizing the porcine acellular dermis and/or the porcine acellular bladder;

step S3, grinding the lyophilized porcine acellular dermis and/or lyophilizing the porcine acellular bladder into fine particles;

step S4, adding the fine particles into the pepsin digestive juice, and stirring for 48-72h to form an extracellular matrix support; and

and step S5, adjusting the pH value of the pepsin digestive juice to inactivate the pepsin, and respectively recombining the proteins in the extracellular matrix scaffold to form the hydrogel.