CN103120807B - Preparation method of ice-induced microstructure soft tissue - Google Patents

Abstract

The invention discloses a method for preparing ice-induced microstructure soft tissue, belonging to the field of biological tissue engineering. In this method, a model with channel network microstructure of soft tissue is firstly designed, and then a resin mold with channel microstructure is prepared by additive manufacturing technology, and pure water is poured into the resin mold to freeze and then peeled off to obtain a model with channel microstructure. Bingzhi mold; finally, pour the pre-configured PVA hydrogel with tissue living cells, growth factors, etc. into the Bingzhi mold system at low temperature, and place it in an environment of 5~10°C after it is stably formed Then, until the ice-induced channel is slowly melted completely, the soft tissue with channel microstructure is finally obtained. This method ensures that the channel structure will not undergo structural deformation due to external effects before forming, and avoids contamination of the original body tissue due to high temperature treatment; the preparation process is relatively simple, easy to operate, and the preparation cost is also very low, and has wide applicability .

Description

A preparation method of ice-induced microstructure soft tissue

Technical field:

The invention relates to a preparation method of artificial organ soft tissue used for organ replacement in the body. The method is a preparation method of tissue engineering three-dimensional soft tissue and belongs to the technical field of bioengineering. the

Background technique:

There are tens of millions of patients suffering from tissue defect or organ failure every year in the world, and about 8 million of these patients are treated with surgery every year in the United States alone. However, the most effective way to solve this problem is artificial replacement of defective organs. There are two main approaches to artificially replace organisms: one is to use autologous or allogeneic organ transplantation, however, the shortage and rejection of donors have always restricted tissue and organ transplantation; the other is to use artificial organs. Traditional artificial organs are usually made of metal, ceramic or polymer materials. Due to the incompleteness of the internal structure, biological activity and degradability of this artificial organ, the replacement effect is not satisfactory and cannot prevent further deterioration of the patient's condition. In order to overcome these limitations, tissue engineering provides the possibility to culture living tissues and organs in vitro, which is expected to restore more biochemical functions of human organs and tissues. Based on this purpose, a preparation method of ice-induced microstructure soft scaffolds is proposed. the

Since different organs have specific internal microstructures, the preparation of organ soft scaffolds with different specific tissue structures according to individual differences is an innovation and exploration of the existing artificial organ preparation. Invention patent CN102525688A discloses a method for manufacturing tissue engineering scaffolds with both internal microstructure and personalized shape. The method first uses a three-dimensional wax-type printer to print the negative of the porous structure; Biological material solution or saline is uniformly made into a slurry, poured into the holes of the negative shape of the porous structure scaffold, cooled and solidified, and the excess biological material on the surface of the scaffold is scraped off; finally, the negative scaffold after perfusion is placed in a heating furnace Heating to a temperature higher than the melting point of the stent wax, continuing to heat until the paraffin melts and disappears, and flushing it with saline, so as to obtain a tissue engineering stent with both internal microstructure and personalized shape. the

The method has the advantages of wide biomaterial adaptability, controllable internal microstructure, and the preparation of tissue engineering scaffolds with both internal microstructure and individualized shape. However, this method still has the following problems and deficiencies: First, it is necessary to design and manufacture the negative structure of the scaffold, which prolongs the production time of the scaffold and increases the cost; secondly, the scaffold needs to be treated at high temperature, which invisibly increases the probability of contamination of the biological scaffold ; Finally, this method cannot be used to prepare soft scaffolds for artificial organs. the

The invention patent with the publication number CN101219240A discloses a method for preparing living tissue with channels. The method firstly dissolves the elastic polymer material in an organic solvent, and adds the cell matrix material into the cell cryopreservation solution; mixes the selected cells with the The cell matrix material solution of the cryopreservation solution is mixed evenly; the living tissue model with pipeline is designed by computer, and according to the discrete accumulation-principle, the above-mentioned elastic polymer material solution, cell- The mixture of matrix material solution is sprayed out through different nozzles to form a hybrid of elastic polymer material with pipeline and cell matrix material. Then refrigerated at low temperature and stored in liquid nitrogen for a long time. After recovery, it can be directly used for the repair of tissues or organs. the

The advantage of this method is that it effectively avoids necrosis of cells if they are not used in time after assembly, and at the same time, this method also overcomes the defect of poor mechanical properties of the assembled cell-material three-dimensional structure. However, this method still has the following problems and deficiencies: the internal three-dimensional channel structure of the living tissue prepared by this method cannot be completely satisfied, because it cannot bear the load before the internal channel structure is finalized, so the actual preparation There is still a big difference between the channel structure in the process and the pre-designed three-dimensional channel structure; in addition, there is still a certain degree of difficulty in direct forming of the channel, and the internal channel of the soft support has certain requirements for the channel size. the

Invention content:

In order to overcome the requirement that the prior art cannot prepare soft organ tissues with specific internal microstructures, the present invention proposes a method for preparing soft tissue scaffolds with ice-induced microstructures. the

The technical solution adopted in the present invention is: a preparation method of ice-induced microstructure soft tissue, first using a computer to design a model with a network microstructure of soft tissue channels, and then using additive manufacturing technology to prepare a resin mold with a channel microstructure , and pour pure water into the resin mold to freeze and then peel off, so as to obtain the ice-induced mold with channel microstructure; finally, pour the pre-configured PVA with tissue living cells, growth factors, etc. into the ice-induced mold system at low temperature After the hydrogel is stably formed, it is placed in an environment of 5-10°C until the ice-induced channels are slowly melted completely, and the finally obtained soft scaffold is a soft tissue scaffold with a microchannel structure. the

The method specifically includes the following steps:

Step 1. Use the computer to design a model with a network microstructure of soft tissue channels;

Step 2. Input the model data built in step 1 into the rapid prototyping machine, and use rapid prototyping technology to manufacture a resin mold with a channel microstructure;

Step 3. Fill the resin mold obtained in step 2 with pure water, and freeze it at a low temperature of -5 to -10°C, then peel off the resin mold to obtain an ice-induced mold with a channel microstructure;

Step 4. Take the organ tissue cells of the individual patient for culture. The cell culture medium is high-sugar DMEM culture medium, wherein 10ml of fetal bovine serum, 29.2mg of glutamine and 10mg of penicillin/streptomycin are added to each 100ml of culture medium. , after several passages to reach the required number, digest with trypsin before use, and add DMEM cell culture medium to prepare a cell suspension with a specific cell density;

Step 5. Take the PVA aqueous solution with a specific PVA concentration according to clinical needs, and uniformly mix the solution with the tissue cell suspension obtained in step 4 according to a certain volume ratio, and control the temperature at 0°C;

Step 6. Mix the mixed solution obtained in step 5 with an appropriate amount of 5% sodium triphosphate aqueous solution to make the hydrogel cross-linked, and immediately pour the mixed solution into the step at a low temperature of -5°C. 3. In the obtained ice-induced mold with channel microstructure, take it out after the PVA hydrogel containing tissue cells is formed stably, and place it in an environment of 5°C until the ice-induced channel is slowly melted completely, and the water flows from The channel structure drains the soft body tissue; the final ice-induced microstructure soft body tissue is prepared. the

The beneficial effects of the present invention are:

1) In the soft tissue preparation process of the present invention, ice is used to prepare the soft tissue channel microstructure, which not only ensures that the channel structure will not undergo structural deformation due to external effects before forming, but also does not need to do any special removal of the negative structure. For processing, you only need to wait for the ice-induced channel to melt in an environment of 5°C, which virtually avoids contamination of the original tissue due to high-temperature processing;

2) The channel microstructure of the present invention can be designed according to actual needs, thereby realizing the artificial control of the tissue channel microstructure, which brings great convenience for practical preparation; this method not only meets the needs of the organization for the channel microstructure, Moreover, its preparation process is relatively simple, easy to operate, the preparation cost is also very low, and has wide applicability;

3) The soft tissue prepared by the organ's own tissue cells and PVA hydrogel used in the present invention, so the soft tissue is a kind of living tissue, can reduce the rejection immune reaction and the pain caused to the patient in the process of organ tissue replacement. the

Description of drawings

Fig. 1 is the hepatic parenchyma scaffold prepared by Bingzhi mold in the embodiment. the

Specific implementation examples

In this embodiment, taking liver soft tissue as an example of preparation, a method for preparing soft tissue with ice-induced microstructure is described, which specifically includes the following steps:

Step 1. Use CAD software to design the tissue model that needs to be prepared and has the network microstructure of the soft tissue tissue channel;

Step 2. Input the model data built in step 1 into the rapid prototyping machine, and use rapid prototyping technology to manufacture a resin mold with a channel microstructure;

Step 3. Fill the resin mold obtained in step 2 with pure water, and peel off the resin mold after freezing at -5°C to obtain an ice-induced mold with channel microstructure;

Step 4. Take 1.0× ¹⁰⁶ liver tissue cells from individual patients for culture. The cell culture medium is high-sugar DMEM culture medium, and 10ml of fetal bovine serum, 29.2mg of glutamine and 10mg of penicillin are added to each 100ml of culture medium. Streptomycin, which has reached the required quantity after multiple passages, was digested with trypsin before use, and added to DMEM cell culture medium to prepare a cell suspension with a cell density of 7.0×10 ⁸ cells/ml;

Step 5. In this embodiment, according to clinical needs, an aqueous solution with a PVA percentage of 25% is taken, and it is uniformly mixed with the tissue cell suspension obtained in step 4 at a volume ratio of 2:3;

Step 6. Mix the mixed solution obtained in step 5 with 5% sodium triphosphate aqueous solution evenly, and the volume ratio is 49:1, so that the hydrogel can be cross-linked, and under the low temperature environment condition of -5°C, Immediately pour the mixed solution into the ice-induced mold with channel microstructure obtained in step 3 until it is full, take it out after the PVA hydrogel containing tissue cells is stably formed, and place it in an environment of 5°C until The ice-induced channels are slowly and completely melted, and the water is discharged from the channel structure; the final ice-induced microstructure soft tissue is obtained. the

Claims (1)

1. ice causes a preparation method for micro structure soft tissue, it is characterized in that, comprises the following steps:

Step 1, design the model with soft tissue channel net micro structure;

Step 2, the model data constructed by step 1 is input to rapidform machine, and utilizes rapid shaping technique manufacture to have the resin die of passage micro structure;

Step 3, in the resin die of step 2 gained the full pure water of perfusion, and carry out under-5 ~-10 DEG C of low temperature environments freezing after, resin die is peeled off, thus the ice obtaining passage micro structure causes mould;

Step 4, the organ-tissue cell of taking sufferer individuality are cultivated, cell culture fluid is DMEM in high glucose culture fluid, wherein be added with 10ml hyclone, 29.2mg glutamine and 10mg penicillin or streptomycin in every 100ml culture fluid, quantity required is reached through repeatedly going down to posterity, use front trypsinization, and add DMEM cell culture fluid thus prepare the cell suspending liquid with specific cells density;

Step 5, according to clinical demand, get the PVA aqueous solution that PVA percentage composition is 25%, and the histiocyte suspension this solution and step 4 obtained is by the volume ratio Homogeneous phase mixing of 2:3, temperature controls at 0 DEG C;

The Sodium triphosphate aqueous solution of step 6, mixed liquor step 5 obtained and 5% concentration is even, volume ratio is 49:1, crosslinked to make hydrogel occur, and under the cryogenic conditions of-5 DEG C, immediately mixed solution being filled into the ice with passage micro structure that step 3 obtains causes in mould, is taken out after stablizing molding containing histiocytic PVA hydrogel, and under being placed in the environment of 5 DEG C, slowly dissolve completely until ice causes passage, water discharges soft tissue from channel design; Prepare final ice and cause micro structure soft tissue.