Three-dimensional scaffold for tumor cell culture and preparation method thereof

Abstract

The invention belongs to the technical field of cells, and particularly relates to a three-dimensional scaffold for tumor cell culture and a preparation method thereof.The preparation method comprises the steps that S1, pretreatment is conducted, S2, enzymolysis is conducted, pigskin obtained in the step S1 is dissolved with pepsin, S3, salting-out is conducted, salting-out is conducted on enzymatic hydrolysate obtained in the step S2, a crude collagen product is obtained, S4, purification is conducted, and the three-dimensional scaffold for tumor cell culture is obtained. S3, purifying the collagen crude product obtained in the step S3 to obtain a purified collagen sample, S5, cross-linking, and performing cross-linking treatment on the freeze-dried collagen scaffold obtained in the step S4 by adopting a chemical cross-linking agent to obtain the required 3D scaffold, so that a three-dimensional scaffold structure meeting production requirements can be provided for cell culture, an in-vivo culture environment can be simulated, and the production efficiency can be improved. The three-dimensional collagen scaffold prepared from collagen derived from pigskin is firm in structure and good in toughness after cross-linking, tumor cells can smoothly migrate into the scaffold, distribution is uniform, and good biocompatibility is shown.

Description

Technical Field

[0001] The invention relates to the technical field of cell culture, in particular to a three-dimensional scaffold for tumor cell culture and a preparation method thereof. Background Art

[0002] Three-dimensional scaffold cell culture has become a hot topic in the field of tumor biology research in recent years. It can establish the connection between cells and between cells and extracellular matrix in vitro, highly restore the real tumor microenvironment in vivo, and has great significance for in vitro tumor model reconstruction. The continuous improvement and development of this technology has made it play a great role in the research of tumor biological behavior, drug screening efficacy analysis, toxicology experiments and targeted therapy.

[0003] Three-dimensional cell culture technology has played a huge role in the study of in vitro cell and tissue culture. It can highly simulate the in vivo growth microenvironment in terms of material and spatial structure, and the cell culture process is more intuitive and the culture conditions are controllable. Continuously exploring, improving and developing new, degradable, and highly plastic scaffold materials, stable spatial structure, excellent mechanical properties and better biocompatibility are necessary requirements for the improvement of three-dimensional cell culture scaffolds.

[0004] Mammalian collagen can be directly decomposed into various amino acids in the body, which can be recycled for cell growth. It is safe, non-toxic, non-polluting, non-irritating, and does not contain allergens. It has good biocompatibility, biodegradability, and resistance. Collagen is different from general double-helix proteins in that it has a triple helix structure. Its three polypeptide chains are left-handed to form a left-handed helix structure, and then they are interlocked through hydrogen bonds to form a strong right-handed superhelix structure. This makes the collagen molecule structure more stable and not easy to break. Pig skin contains a lot of collagen, which is cheap and easy to obtain, making it feasible as a raw material for three-dimensional scaffold materials.

[0005] Therefore, the present invention intends to combine the advantages of the source of pig skin collagen to prepare a three-dimensional cell culture scaffold for CHO cell culture and study its cell culture effect. Summary of the invention

[0006] The purpose of this section is to summarize some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0007] Therefore, the purpose of the present invention is to provide a method for preparing a three-dimensional scaffold for tumor cell culture. The three-dimensional cell culture scaffold prepared by the present invention has a porous structure inside the collagen scaffold, and the pore diameter is 30 to 150 μm. For CHO cell culture, the cells migrate smoothly into the scaffold and are evenly distributed. The scaffold can provide an active surface for cell migration and adhesion, and can simulate the three-dimensional environment in the body to the greatest extent, so that the characteristics of cells cultured in vitro are not significantly different from those of cells in vivo. In order to solve the above technical problems, according to one aspect of the present invention, the present invention provides the following technical solutions:

[0008] A method for preparing a three-dimensional scaffold for tumor cell culture, comprising:

[0009] S1 pretreatment, take fresh pig skin, remove hair and cut into 2mm*2mm square pieces, add degreasing solution to degrease, and wash until the pH value is neutral;

[0010] S2 enzymatic hydrolysis, dissolving the pigskin obtained in step S1 with pepsin;

[0011] S3 salting out, salting out the enzymatic hydrolysate obtained in step S2 to obtain a crude collagen product;

[0012] S4 purification, purifying the crude collagen product obtained in step S3 to obtain a purified collagen sample;

[0013] S5 cross-linking, using a chemical cross-linking agent to cross-link the freeze-dried protein scaffold obtained in step S4 to obtain the desired 3D scaffold.

[0014] As a preferred embodiment of the method for preparing a three-dimensional scaffold for tumor cell culture described in the present invention, in step S2, in order to ensure the collagen content in the protein extracted during the enzymatic hydrolysis process, the pH of the solution is precisely adjusted with acetic acid, and the pH of the solution is accurately measured with a pH meter to maintain the pH at 2.0.

[0015] As a preferred embodiment of the method for preparing a three-dimensional scaffold for tumor cell culture described in the present invention, the specific operation of step S3 is to filter the enzymatic hydrolyzate obtained in step S2, collect the filtrate, and dropwise add 2.5 mol / L sodium hydroxide solution, adjust the pH of the filtrate until the filtrate becomes neutral or weakly alkaline, add sodium chloride to make the final concentration of sodium chloride in the solution 4 mol / L, put it in a refrigerator, salt out at 4°C for 12 hours, centrifuge the product after salting out, and the obtained precipitate is the crude collagen product.

[0016] As a preferred embodiment of the method for preparing a three-dimensional scaffold for tumor cell culture described in the present invention, the specific operation of step S4 is to place the crude collagen product obtained in step S3 in a clean beaker, fully dissolve it with a small amount of acetic acid solution with a pH of 2.5, centrifuge, collect the supernatant, and then pour the obtained supernatant into a dialysis bag with a retention capacity of 8000-14000 kDa, dialyze in acetic acid solution with a pH of 3.0 for 2 days, change the solution every 5 to 6 hours, and then dialyze with distilled water for 1 day, change the water every 2 hours. After the dialysis is completed, the solution in the dialysis bag is placed in a culture dish and sealed for storage, placed in an ultra-low temperature freezer for pre-freezing for 2 hours, and then immediately transferred to a freeze dryer for freeze drying until freeze-dried, so as to obtain a purified collagen sample.

[0017] As a preferred embodiment of the method for preparing a three-dimensional scaffold for tumor cell culture described in the present invention, in order to avoid adding a large amount of sodium chloride solid at one time, which may cause a large amount of collagen to precipitate locally and thus affect the quality of the final collagen product, the sodium chloride solution should be added dropwise.

[0018] As a preferred embodiment of the method for preparing a three-dimensional scaffold for tumor cell culture described in the present invention, the specific operation of step S5 is to take out the freeze-dried protein scaffold obtained in step S4, place it in a beaker, pour 2 mL of ethanol solution with a volume fraction of 0.4, then accurately weigh the cross-linking agent and place it in the beaker, cross-link at room temperature for 6 hours, after the cross-linking is completed, add an appropriate amount of 0.1 mol / L disodium hydrogen phosphate solution to the beaker, place it at room temperature for 2 hours to fully remove the cross-linking agent, and then wash the protein with 40% volume fraction ethanol, wash it 4 times in 2 hours, and then wash the collagen with distilled water until it is neutral, start centrifugation, pour off the supernatant, take the lower layer of collagen, put the collagen into a 24-well plate, put an equal amount of collagen in each well plate, so that the collagen is flat and there are no bubbles, put it into a freezer for pre-freezing for 2 hours, and then immediately transfer it to a freeze dryer to obtain the required 3D scaffold.

[0019] As a preferred embodiment of the method for preparing a three-dimensional scaffold for tumor cell culture described in the present invention, wherein: in the step S5, during the cross-linking process, carbodiimide + 2-morpholineethanesulfonic acid + N-hydroxysuccinimide are used as chemical cross-linking agents, and the masses of each are 0.0191g carbodiimide, 0.0195g 2-morpholineethanesulfonic acid and 0.0115g N-hydroxysuccinimide respectively.

[0020] The three-dimensional cell culture scaffold for tumor cell culture prepared by any of the methods described above is characterized in that the interior of the collagen scaffold is observed to be porous from a scanning electron microscope (SEM) image, with a pore diameter of 30 to 150 μm.

[0021] As a preferred embodiment of the three-dimensional cell culture scaffold for tumor cell culture described in the present invention, the cells migrate smoothly into the scaffold and are evenly distributed.

[0022] Compared with the existing technology: Since pig skin collagen has good biocompatibility, blood compatibility and safety, a scaffold material with a porous structure can be obtained. The porous scaffold material is conducive to cell adhesion and growth, and provides support for cell growth. It is used as a scaffold for CHO cell culture, which can simulate the three-dimensional culture environment in the body. Compared with the two-dimensional culture method, the cell migration is more efficient and the distribution is more uniform. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the present invention will be described in detail below in combination with the accompanying drawings and detailed embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0024] Figure 1 This is a macroscopic image of the uncrosslinked scaffold of the present invention;

[0025] Figure 2 This is a macroscopic image of the cross-linked stent of the present invention;

[0026] Figure 3 This is a SEM image of the cross-linked stent of the present invention;

[0027] Figure 4 It is the infrared spectrum of the present invention;

[0028] Figure 5 (a) HE staining schematic diagram, (b) DiI fluorescence observation schematic diagram of the present invention;

[0029] Figure 6 This is a schematic diagram showing the effect of the scaffold extract of the present invention on the viability of CHO cells;

[0030] Figure 7 These are cells that grow adherently under normal conditions in the two-dimensional culture of the present invention (control group). DETAILED DESCRIPTION

[0031] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0032] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0033] Secondly, the present invention is described in detail with reference to schematic diagrams. When describing the embodiments of the present invention in detail, for the sake of convenience, the cross-sectional diagrams showing the device structure will not be partially enlarged according to the general scale, and the schematic diagrams are only examples, which should not limit the scope of protection of the present invention. In addition, in actual production, the three-dimensional dimensions of length, width and depth should be included.

[0034] To make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0035] The present invention provides a three-dimensional scaffold for tumor cell culture and a preparation method thereof, which can provide a three-dimensional scaffold structure that meets production requirements for cell culture, simulate the in vivo culture environment, and improve the in vitro culture effect of cells.

[0036] Step S1, pre-treating fresh pigskin to obtain depilated and defatted pigskin in square pieces of 2 mm×2 mm.

[0037] Step S2, dissolving the pig skin in step 1) with pepsin.

[0038] In order to ensure the collagen content in the protein extracted during the enzymatic hydrolysis process, the pH of the solution must be precisely adjusted with acetic acid, and the pH of the solution must be accurately measured with a pH meter to maintain pH = 2.0.

[0039] Step S3, salting out the enzymatic hydrolyzate obtained in step S2 to obtain a crude collagen product.

[0040] During the salting out process, sodium chloride solution should be added dropwise to avoid adding a large amount of sodium chloride solid at one time, which may cause local precipitation of collagen and affect the quality of the final collagen product.

[0041] Step S4, purifying the crude collagen product obtained in step S3 to obtain a purified collagen sample.

[0042] Step S5, cross-linking the freeze-dried protein scaffold obtained in step S4 using a chemical cross-linking agent to obtain the desired 3D scaffold.

[0043] The freeze-dried protein scaffold was taken out and placed in a beaker. 2 mL of ethanol solution with a volume fraction of 0.4 was poured into it. Then 0.0191 g of carbodiimide, 0.0195 g of 2-morpholineethanesulfonic acid and 0.0115 g of N-hydroxysuccinimide were accurately weighed and placed in the beaker. The mixture was cross-linked at room temperature for 6 hours.

[0044] Furthermore, after the cross-linking is completed, add an appropriate amount of 0.1 mol / L disodium hydrogen phosphate solution to the beaker and leave it at room temperature for 2 hours to fully remove the cross-linking agent. Then wash the protein with 40% ethanol by volume, wash it 4 times in 2 hours, and then wash the collagen with distilled water until it is neutral, start centrifugation, pour off the supernatant, and take the lower layer of collagen. Put the collagen in a 24-well plate, put an equal amount of collagen in each well plate, so that the collagen is flat and there are no bubbles. Put it in a freezer for pre-freezing for 2 hours, and then immediately transfer it to a freeze dryer to finally obtain the desired 3D scaffold.

[0045] The process of introducing cells into the 3D scaffold culture includes the following steps:

[0046] 1) Preparation before the experiment

[0047] This experiment uses tumor cells (CHO), and the cells must be cultured under sterile conditions to prevent contamination. Therefore, before the experiment, sterilization preparations must be made, and the DMEM culture medium and PBS solution required for cell culture must be prepared in advance and stored in a refrigerator at 4°C.

[0048] 2) Cell recovery

[0049] Heat the DMEM culture medium in a 37°C water bath. After heating evenly, take out the cryotube from the ultra-low temperature storage refrigerator and heat it in a 37°C water bath. After it is completely thawed, quickly transfer it to the clean bench, pour the cell solution into a sterilized 10mL centrifuge tube, add 6mL EMDM culture medium, shake it gently and evenly, adjust the centrifuge speed to 1500, and after 5 minutes, do not shake when taking the centrifuge tube, transfer it to the clean bench, use a 5mL pipette to transfer the upper 6mL to the waste liquid tank from top to bottom, and transfer the lower two milliliters evenly to two disposable culture dishes, change a 5mL new pipette tip, and then add 4mL EMDM culture medium to the two culture dishes respectively, and shake the culture dishes carefully to disperse the cells. After observation under a microscope, transfer it to a 37°C, 5% carbon dioxide constant temperature incubator for in vitro culture, and subculture and collect cells according to previously reported methods.

[0050] 3) Cell 3D scaffold culture and fluorescence observation

[0051] Take 2mL of 10mg / LDiI working solution and add it to the collected cells to resuspend the cells. Incubate at 37℃ and 4℃ for 10min in sequence under light-proof conditions. Then adjust the centrifuge speed to 1500. After running for 10min, rinse the cells twice with 2mL PBS and adjust the cell density to 2×106 cells / mL. Then place the three-dimensional collagen scaffold in a disposable culture dish, take 100μL CHO cell suspension and place it on the surface of the scaffold. Let it stand for 1 hour, then add 900μL DMEM culture medium and culture it in a 37℃, 5% carbon dioxide incubator.

[0052] (3.1) Immerse the three-dimensional collagen scaffold in 75% alcohol for sterilization. When the scaffold sinks to the bottom of the alcohol solution, remove the scaffold, transfer it to a clean bench to dry naturally, and store it sterilely at 4°C for later use.

[0053] (3.2) The cells adhered to the wall and grew. When the confluence reached 80% to 90%, the cells were collected and the cell suspension was centrifuged at 1000 rpm for 10 minutes to remove the cell culture medium.

[0054] (3.3) Add 2 mL of 10 mg / L DiI working solution to resuspend the cells, incubate at 37°C and 4°C in the dark for 10 min each, centrifuge the cell suspension at 1000 rpm for 10 min, collect the cells and rinse twice with 2 mL of PBS, and adjust the cell density to 2 × 106 cells / mL using cell culture medium.

[0055] (3.4) Place the three-dimensional collagen scaffold in a 24-well plate, take 100 μL of cell suspension and inoculate it on the surface of the scaffold. After standing for 1 hour, add 900 μL of cell culture medium and culture it in a 37°C, 5% CO2 incubator for 3 days.

[0056] (3.5) The cell culture scaffold was removed, immersed in 4% paraformaldehyde and fixed at 4°C for 24 hours, and then transferred to 20% sucrose solution for dehydration at 4°C until the scaffold sank to the bottom of the solution. The dehydrated cell scaffold was embedded in OCT cryogel and frozen at -20°C with a thickness of 10 μm. After the sections were adhered with anti-shedding carrier sheets, the growth of cells in the scaffold was observed under a fluorescence microscope and microscopic photography was performed. The fluorescence microscope used green excitation light, the cells emitted orange-red fluorescence, and the scaffold network fibers emitted red fluorescence.

[0057] Although the present invention has been described above with reference to the embodiments, various modifications may be made thereto and parts thereof may be replaced by equivalents without departing from the scope of the present invention. In particular, as long as there is no structural conflict, the various features in the embodiments disclosed in the present invention may be used in combination with each other in any manner, and the fact that these combinations are not exhaustively described in this specification is only for the sake of omitting space and saving resources. Therefore, the present invention is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. A method for preparing a three-dimensional scaffold for tumor cell culture, characterized in that: The method comprises the following steps: S1 pretreatment, take fresh pig skin, remove hair and cut into 2mm*2mm square pieces, add degreasing solution to degrease, and wash until the pH value is neutral; S2 enzymatic hydrolysis, dissolving the pigskin obtained in step S1 with pepsin; S3 salting out, salting out the enzymatic hydrolysate obtained in step S2 to obtain a crude collagen product; S4 purification, purifying the crude collagen product obtained in step S3 to obtain a purified collagen sample; S5 cross-linking, using a chemical cross-linking agent to cross-link the freeze-dried protein scaffold obtained in step S4 to obtain the desired 3D scaffold.

2. The method for preparing a three-dimensional scaffold for tumor cell culture according to claim 1, characterized in that: In step S2, in order to ensure the collagen content in the protein extracted during the enzymatic hydrolysis process, the pH of the solution is precisely adjusted with acetic acid, and the pH of the solution is accurately measured with a pH meter to maintain the pH at 2.

0.

3. The method for preparing a three-dimensional scaffold for tumor cell culture according to claim 1, characterized in that: The specific operation of step S3 is to filter the enzymatic hydrolyzate obtained in step S2, collect the filtrate, and drop 2.5 mol / L sodium hydroxide solution to adjust the pH of the filtrate until the filtrate becomes neutral or weakly alkaline, add sodium chloride to make the final concentration of sodium chloride in the solution 4 mol / L, put it in a refrigerator, salt out at 4°C for 12 hours, centrifuge the product after salting out, and the obtained precipitate is the crude collagen product.

4. The method for preparing a three-dimensional scaffold for tumor cell culture according to claim 1, characterized in that: The specific operation of step S4 is to place the crude collagen product obtained in step S3 in a clean beaker, fully dissolve it with a small amount of acetic acid solution with a pH of 2.5, centrifuge, collect the supernatant, and then pour the obtained supernatant into a dialysis bag with a cutoff of 8000-14000 kDa, dialyze in acetic acid solution with a pH of 3.0 for 2 days, change the solution every 5 to 6 hours, and then dialyze with distilled water for 1 day, change the water every 2 hours. After the dialysis is completed, place the solution in the dialysis bag in a culture dish and seal it for storage, put it in an ultra-low temperature freezer for pre-freezing for 2 hours, and then immediately transfer it to a freeze dryer for freeze drying until freeze-drying, so as to obtain a purified collagen sample.

5. The method for preparing a three-dimensional scaffold for tumor cell culture according to claim 3, characterized in that: In order to avoid adding a large amount of sodium chloride solid at one time, which may cause local precipitation of collagen and affect the quality of the final collagen product, the sodium chloride solution should be added drop by drop.

6. The method for preparing a three-dimensional scaffold for tumor cell culture according to claim 1, characterized in that: The specific operation of step S5 is to take out the freeze-dried protein scaffold obtained in step S4, place it in a beaker, pour 2 mL of ethanol solution with a volume fraction of 0.4, then accurately weigh the cross-linking agent and place it in the beaker, cross-link at room temperature for 6 hours, and after the cross-linking is completed, add an appropriate amount of 0.1 mol / L disodium hydrogen phosphate solution to the beaker, leave it at room temperature for 2 hours to fully remove the cross-linking agent, and then wash the protein with 40% volume fraction ethanol, wash it 4 times in 2 hours, and then wash the collagen with distilled water until it is neutral, start centrifugation, pour off the supernatant, take the lower layer of collagen, put the collagen into a 24-well plate, put an equal amount of collagen in each well plate, so that the collagen is flat and there are no bubbles, put it into a freezer for pre-freezing for 2 hours, and then immediately transfer it to a freeze dryer to obtain the desired 3D scaffold.

7. The method for preparing a three-dimensional scaffold for tumor cell culture according to claim 6, characterized in that: In the step S5, during the cross-linking process, carbodiimide + 2-morpholineethanesulfonic acid + N-hydroxysuccinimide are used as chemical cross-linking agents, and the masses of each are 0.0191 g of carbodiimide, 0.0195 g of 2-morpholineethanesulfonic acid and 0.0115 g of N-hydroxysuccinimide.

8. A three-dimensional cell culture scaffold for tumor cell culture prepared by the method according to any one of claims 1 to 7, characterized in that: From the scanning electron microscope (SEM) image, it can be observed that the interior of the collagen scaffold is porous, with a pore diameter of 30 to 150 μm.

9. The three-dimensional cell culture scaffold for tumor cell culture according to claim 8, characterized in that: The cells migrated smoothly into the scaffold and were evenly distributed.

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