阅读说明：本技术 用于组织修复的支架的低温冷冻方法 (Cryogenic freezing method of scaffolds for tissue repair ) 是由 章毅 蔡海波 王进 伍婷 陈亮 于 2021-10-11 设计创作，主要内容包括：一种用于组织修复的支架的低温冷冻方法,用于组织修复的支架与间充质干细胞在24孔培养板中培养；每2天换液,培养7天后,形成结构良好的组织工程结构；将制得的组织工程结构浸没在冷冻保护剂中,并在培养箱中孵育12小时；用于组织修复的支架由没食子酸接枝壳聚糖制成。本发明的低温冷冻方法,适用于含活细胞的组织工程结构(如：支架)的低温冷冻保存,将细胞的存活率和复苏率维持在较高水平,并能使冷冻的干细胞在复苏后继续保持干性和分化潜能。(A low-temperature freezing method of a scaffold for tissue repair, the scaffold for tissue repair is cultured with mesenchymal stem cells in a 24-well culture plate; changing the liquid every 2 days, and culturing for 7 days to form a tissue engineering structure with a good structure; immersing the prepared tissue engineering structure in a cryoprotectant, and incubating for 12 hours in an incubator; the scaffold for tissue repair is made of gallic acid grafted chitosan. The low-temperature freezing method is suitable for low-temperature freezing preservation of tissue engineering structures (such as scaffolds) containing living cells, maintains the survival rate and recovery rate of the cells at a higher level, and can enable the frozen stem cells to continue to maintain the dryness and differentiation potential after recovery.)

1. A cryogenic freezing method, comprising:

culturing the scaffold for tissue repair and mesenchymal stem cells in a 24-well culture plate; changing the liquid every 2 days, and culturing for 7 days to form a tissue engineering structure with a good structure;

immersing the prepared tissue engineering structure in a cryoprotectant, and incubating for 12 hours in an incubator;

the scaffold for tissue repair is prepared from gallic acid grafted chitosan.

2. The cryogenic freezing method according to claim 1, wherein the scaffold for tissue repair is immersed in 70% v/v ethanol overnight before being co-cultured with mesenchymal stem cells, and then irradiated under ultraviolet rays for 12 hours.

3. Cryogenic freezing process according to claim 1, wherein said cryoprotectant comprises a trehalose derivative comprising trehalose and epichlorohydrin, said trehalose and said epichlorohydrin being covalently bound.

4. A method of cryogenic freezing as claimed in claim 3, wherein the trehalose derivative is present in an amount of 25mg/mL to 100 mg/mL.

5. A method of cryogenic freezing as claimed in claim 3, wherein the trehalose derivative is present in an amount of 50 mg/mL.

6. A low-temperature freezing method as claimed in claim 3, wherein the trehalose derivative comprises 4-12 parts by weight of trehalose and 3-10 parts by weight of epichlorohydrin.

7. The trehalose derivatives according to claim 3, wherein the trehalose derivatives are obtained by the following method

Dissolving 4-12 parts by weight of trehalose and NaOH in deionized water, reacting and stirring at 65-75 ℃ for 15-45 minutes at a pH of 11-12 to obtain a transparent viscous liquid;

and then adding 3-10 parts by weight of epoxy chloropropane, standing for 36-72 hours, adjusting the pH value of the solution to be neutral by using hydrochloric acid, dialyzing in deionized water for 3-5 days, and freeze-drying to obtain the trehalose derivative.

8. The cryoprotectant of claim 1, further comprising fetal bovine serum and DMEM media.

9. The cryoprotectant of claim 8, further comprising 10% v/v fetal bovine serum.

10. The cryoprotectant of claim 8, further comprising 90% v/v DMEM medium.

Technical Field

The present invention relates to a method for preserving biological products, in particular to a method for freezing scaffolds for tissue repair at low temperature, and the biological activity of the biological products (such as cells) in the scaffolds is maintained.

Background

Tissue engineering and regenerative medicine are considered to be a promising approach to repair damaged tissues and maintain biological function. Among the therapeutic methods of tissue engineering and regenerative medicine, cell-based therapeutic methods are one of the most recent hot spots. These therapies have been focused on the ability to regenerate damaged tissues and provide a number of beneficial cytokines. In particular, stem cells can be maintained dry by implanting them in a scaffold. In previous studies, we established tissue engineering structures based on gallic acid grafted chitosan scaffolds and mesenchymal stem cells. In addition, the difficulty in scaffold fabrication is the encapsulation of cells into the scaffold to produce an active composite biomaterial; however, the process of procurement, growth and packaging is very cumbersome and time consuming and difficult to apply to acute diseases. Therefore, the development of an effective cryopreservation strategy to maintain the biological activity and function of the tissue engineering structure is one of the most important research fields of modern tissue engineering and regenerative medicine. The integrity tissue engineering structure capable of being preserved not only can save the time of donors and recipients, but also can establish a global high-quality tissue engineering structure cell biological information base. In preparing engineered tissues for clinical applications, cryopreservation strategies have the potential to enable the off-the-shelf supply of tissue engineered structures.

Disclosure of Invention

It is an object of the present invention to provide a cryogenic freezing method for cryo-preservation of scaffolds for tissue repair.

It is another object of the present invention to provide a cryogenic freezing method for cryo-preservation of scaffolds for tissue repair containing bioactive substances.

A cryogenic freezing method comprising:

scaffolds and mesenchymal stem cells for tissue repair (e.g., 2X 10)⁴Individual cells) were cultured in 24-well culture plates. Changing the liquid every 2 days, and culturing for 7 days to form a tissue engineering structure with a good structure;

the prepared tissue engineering structure was immersed in cryoprotectant and incubated in an incubator for 12 hours.

According to the method, the scaffold for tissue repair is prepared by grafting gallic acid on chitosan. The scaffolds were soaked in 70% v/v ethanol overnight before co-culturing with mesenchymal stem cells, and then irradiated under ultraviolet rays for 12 hours.

The cryoprotectant adopted by the method comprises trehalose derivatives, fetal calf serum and DMEM medium.

The other cryoprotectant comprises 25 mg/mL-100 mg/mL of trehalose derivatives, fetal calf serum and a DMEM medium.

Another cryoprotectant comprises 25 mg/mL-100 mg/mL trehalose derivatives, 10% v/v fetal calf serum and DMEM medium.

Another cryoprotectant comprises 25 mg/mL-100 mg/mL of trehalose derivatives, 10% v/v fetal bovine serum and 90% v/v DMEM medium.

Another cryoprotectant comprises 50mg/mL trehalose derivatives, fetal bovine serum and DMEM medium.

Another cryoprotectant, comprises 50mg/mL trehalose derivatives, 10% v/v fetal bovine serum and DMEM medium.

Another cryoprotectant, comprising 50mg/mL trehalose derivatives, 10% v/v fetal bovine serum, and 90% v/vDMEM medium.

According to the method, the trehalose derivatives comprise trehalose and epichlorohydrin, and the trehalose and the epichlorohydrin are covalently bonded.

The trehalose derivative comprises the following components in parts by weight:

4-12 trehalose and 3-10 epichlorohydrin, wherein the trehalose and the epichlorohydrin are covalently bonded.

The trehalose derivative comprises the following components in parts by weight:

4-12 parts of trehalose and 3-10 parts of epoxy chloropropane, and is prepared by the following method:

dissolving 4-12 parts by weight of trehalose and NaOH in deionized water, reacting and stirring at 65-75 ℃ for 15-45 minutes at a pH of 11-12 to obtain a transparent viscous liquid. Then adding 3-10 parts by weight of epoxy chloropropane, standing for 36-72 hours, adjusting the pH value of the solution to be neutral (such as the pH value of 7 +/-0.2) by using hydrochloric acid, dialyzing in deionized water for 3-5 days, and freeze-drying to obtain the trehalose derivative.

The trehalose derivative can enter cells, and is used as a cryoprotectant for the cells, and the dosage is as follows: but not limited to 25 mg/mL-100 mg/mL, has the effect of protecting frozen cells, improves the survival rate and the recovery rate of the cells, and can keep the dryness and differentiation potential of the stem cells.

The low-temperature freezing method can maintain the survival rate and the recovery rate of the cells at a higher level by the low-temperature freezing preservation of the tissue engineering structure (such as a scaffold) containing the living cells, and can ensure that the frozen stem cells continue to maintain the dryness and differentiation potential after recovery.

Drawings

FIG. 1 shows the survival rate of mesenchymal stem cells in a tissue engineering structure frozen at low temperature;

FIG. 2 is the recovery rate of mesenchymal stem cells in a tissue engineering structure frozen at low temperature;

FIG. 3 shows the proliferation capacity of mesenchymal stem cells in a tissue engineering structure frozen at low temperature;

FIG. 4 is a graph of the colony forming ability of mesenchymal stem cells in a cryogenically frozen tissue-engineered structure;

FIG. 5 is a data statistics chart showing the differentiation capacity of adipose-forming, osteogenic and chondrogenic mesenchymal stem cells in a tissue engineering structure frozen at a low temperature.

Detailed Description

The technical scheme of the invention is described in detail in the following with reference to the accompanying drawings. Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the spirit and scope of the invention as defined in the appended claims.

The tissue engineering structure containing the mesenchymal stem cells adopted by the following embodiments of the invention is prepared by the following method:

culturing scaffold (made of gallic acid grafted chitosan) for tissue repair and mesenchymal stem cells in 24-well culture plate; changing the liquid every 2 days, and culturing for 7 days to form a tissue engineering structure with a good structure;

immersing the prepared tissue engineering structure in a cryoprotectant, and incubating in an incubatorAnd breeding for 12 hours. Specifically, such as: the gallic acid grafted chitosan scaffold was soaked in 70% v/v ethanol overnight and then irradiated under uv for 12 hours. Then, gallic acid grafted chitosan scaffold and mesenchymal stem cells (2 × 10)⁴Individual cells) were cultured in 24-well culture plates. Changing the culture solution every 2 days, and culturing for 7 days to form a tissue engineering structure.

The preparation method of the gallic acid grafted chitosan scaffold comprises the following steps:

first 28mmol of gallic acid and 2.8mmol of EDC were dissolved in 40mL 70% ethanol and 2.8mmol of NHS was added to the solution. The resulting solution was stirred in an ice bath, then 1.5g of chitosan dispersed in 110mL of 70% ethanol was added after 1 hour. The solution was further stirred in an ice bath for 30 minutes and finally at room temperature for 24 hours. The precipitate was collected by filtration and washed with ethanol. After 3 days of dialysis against deionized water to remove possible residual reagents, gallic acid-grafted chitosan was obtained by vacuum freeze-drying. The porous gallic acid grafted chitosan scaffold is prepared by utilizing the physical property that gallic acid grafted chitosan is dissolved in an acid solution and is not dissolved under an alkaline condition. First, 1g of gallic acid-grafted chitosan was dissolved in 50ml of 0.1 v/v% glacial acetic acid. Then 250 mu L of gallic acid grafted chitosan solution is added into each hole of the polytetrafluoroethylene plate, and the mixture is kept in a refrigerator at the temperature of 20 ℃ below zero for 24 hours. The samples were then lyophilized in a freeze dryer. The samples were deacidified with 2 w/v% NaOH solution for 4 hours and lyophilized in a freeze dryer. Finally obtaining the gallic acid grafted chitosan scaffold.

The experimental method for the survival rate of the mesenchymal stem cells in the tissue engineering structure comprises the following steps:

the prepared tissue engineering structure was washed twice with PBS buffer, and the mesenchymal stem cells were digested with 0.25% trypsin and 1mM EDTA. The cells were then centrifuged and the resulting cell suspension, trypan blue 1:1, was mixed and counted on a hemocytometer. The survival rate of the cells was (viable cells/total cells) × 100.

The experimental method for the Survival Rate (Survival Rate,%) and the resuscitation Rate (Cell Recovery Rate,%) of mesenchymal stem cells in the frozen tissue engineering structure is as follows:

the frozen tissue engineering structures were washed twice with PBS buffer. Mesenchymal stem cells were digested with 0.25% trypsin and 1mM EDTA. The cells were then centrifuged. The cell suspensions, trypan blue 1:1 were then mixed and counted on a hemacytometer. The survival rate of the cells (live cells after cryopreservation/total cells after cryopreservation) × 100, and the recovery rate of the cells (live cells after cryopreservation/live cells before cryopreservation) × 100.

The repopulation capacity (Cell Proliferation) of mesenchymal stem cells in a tissue engineering structure is determined by CCK-8. The specific method comprises the following steps: digesting the mesenchymal stem cells from the tissue engineering structure, then inoculating the mesenchymal stem cells into a 24-well cell culture plate at a density of 10,000/well and 5% CO at 37 ℃₂And (5) culturing. The solution was mixed with the solution of CCK-8 in a volume of 9:1 using alpha-MEM to obtain a working solution of CCK-8, 200. mu.L of the working solution of CCK-8 was added and incubated with the cells for 2 hours, and then 100. mu.L of the sample solution was taken out and absorbance was measured at 450 nm.

The experimental method for the dryness and three differentiation capacities of the mesenchymal stem cells in the tissue engineering structure comprises the following steps:

and (3) detecting the forming capability of the mesenchymal stem cell colony in the tissue engineering structure by using a crystal violet staining method. Digesting the mesenchymal stem cells from the tissue engineering structure. Then, the mesenchymal stem cells were inoculated into 6-well cell culture plates at a density of 200 cells/well and 37 ℃ with 5% CO₂The culture was carried out for 14 days. Before crystal violet staining, wash 2 times with PBS and fix with 4% paraformaldehyde for 15 min at room temperature. Add crystal violet staining solution and incubate for 30 min.

Three differentiation potentials of the mesenchymal stem cells on the tissue engineering structure are detected through adipogenesis, osteogenesis and cartilage differentiation. According to the manufacturer's instructions. Adopting a adipogenic culture medium, an osteogenic culture medium and a chondrogenic culture medium to respectively mediate MSCgo^TMThe differentiation capability of the mesenchymal stem cells after the tissue engineering structure is frozen is researched. After 21 days of culture, cells were stained with adipogenic, osteogenic and cartilage staining kit. Washed 2 times with PBS and fixed with 4% paraformaldehyde for 15 minutes at room temperature. Staining the cells with oil Red O (oil Red O), alizarin Red S (Alzarin Red S) and Alisin Blue (Alien Blue), staining the cells withIn adipogenic, osteogenic and cartilaginous differentiation. The cells were then washed three times with wash solution to remove excess dye. The differentiated cells were then observed with an optical microscope. Finally, oil red O, alizarin Red S and Alisin blue stained cells were analyzed for adipogenic, osteogenic and chondrogenic at wavelengths of 500nm, 550nm and 600 nm.

Example 1

The survival rate of mesenchymal stem cells in the tissue engineering structure is shown in figure 1.

The proliferation capacity of mesenchymal stem cells in tissue engineering structures is shown in figure 3.

The colony forming ability of the mesenchymal stem cells in the tissue engineering structure is shown in figure 4, and the three differentiation abilities of the mesenchymal stem cells in the tissue engineering structure are shown in figure 5.

Example 2

The tissue engineering structures were cryopreserved in fetal bovine serum (90% FBS) cryopreserved tubes containing 10% DMSO. The frozen tubes were then stored sequentially at 4 ℃ for 30 minutes, -20 ℃ for 60 minutes, -80 ℃ overnight, and finally under liquid nitrogen for 30 days.

The survival rate of the mesenchymal stem cells in the tissue engineering structure after cryopreservation is shown in fig. 1. The survival rate of the mesenchymal stem cells in the tissue engineering structure after cryopreservation is shown in fig. 2.

The proliferation ability of the mesenchymal stem cells in the tissue engineering structure after freezing storage is shown in figure 3, the colony formation ability of the mesenchymal stem cells is shown in figure 4, and the three differentiation abilities of the mesenchymal stem cells are shown in figure 5.

Example 3 electrochemical analysis of breast cancer Stem cells in the Presence of interfering cells

4.00g trehalose and 1.82g NaOH dissolved in 10mL deionized water, at 75 degrees C reaction and stirring for 30 minutes, get a transparent viscous liquid. Then 3.23g of epichlorohydrin was added. The solution was allowed to stand, causing an increase in viscosity. After 48 hours, the pH of the solution was adjusted to 7.00 with hydrochloric acid. Dialyzed against deionized water for 3 days, and then lyophilized in vacuo to give trehalose polymer.

The prepared tissue engineering structure was immersed in a frozen stock solution of the above-synthesized trehalose derivative (25mg/mL) and incubated in an incubator for 12 hours. Each tissue engineering construct was then transferred to a sterile 1.5mL cryovial and 0.75mL trehalose derivative cryovial (25mg/mL) was added. The frozen tubes were then stored sequentially at 4 ℃ for 30 minutes, -20 ℃ for 60 minutes, -80 ℃ overnight, and finally under liquid nitrogen for 30 days.

The survival rate of the mesenchymal stem cells in the frozen tissue engineering structure is shown in figure 1, and the recovery rate of the mesenchymal stem cells is shown in figure 2.

Example 4

The prepared tissue engineering structure was immersed in a frozen stock solution of trehalose derivative (50mg/mL) prepared in example 3 and incubated in an incubator for 12 hours. Each tissue engineering construct was then transferred to a sterile 1.5mL cryovial and 0.75mL trehalose derivative cryovial (25mg/mL) was added. The frozen tubes were then stored sequentially at 4 ℃ for 30 minutes, -20 ℃ for 60 minutes, -80 ℃ overnight, and finally under liquid nitrogen for 30 days.

The survival rate of the mesenchymal stem cells in the frozen tissue engineering structure is shown in figure 1, and the recovery rate of the mesenchymal stem cells is shown in figure 2.

The proliferation ability of mesenchymal stem cells in the tissue engineering structure after cryopreservation is shown in fig. 3, the colony formation ability of mesenchymal stem cells is shown in fig. 4, and the differentiation ability of adipose, osteogenic and chondrogenic of mesenchymal stem cells is shown in example 4 in fig. 5.

Example 5

The prepared tissue engineering structure was immersed in the frozen stock solution of trehalose derivatives (100mg/mL) of example 3 and incubated in an incubator for 12 hours. Each tissue engineering construct was then manually transferred to a sterile 1.5mL cryovial and 0.75mL trehalose derivative cryovial (25mg/mL) was added. The frozen tubes were then stored sequentially at 4 ℃ for 30 minutes, -20 ℃ for 60 minutes, -80 ℃ overnight, and finally under liquid nitrogen for 30 days.

The survival rate of the mesenchymal stem cells in the frozen tissue engineering structure is shown in figure 1, and the recovery rate of the mesenchymal stem cells is shown in figure 2.

Compared with the above examples, the trehalose derivatives provided by the present application have high cell recovery rate (39.56 ± 2.95% -52.02 ± 4.01%) and survival rate (60.75 ± 5.02% -85.38 ± 2.39%) for tissue engineering scaffolds kept frozen, and mesenchymal stem cells in the frozen tissue engineering scaffolds have desiccation and three differentiation capacities, so that the trehalose derivatives can be used as a substitute for DMSO for cryogenically freezing tissue engineering scaffolds containing cells.

The trehalose derivative with the concentration of 50mg/mL is added into the cryopreservation liquid, so that the cell recovery rate and the cell survival rate of the cells can be maintained at high levels, and the economic benefit is better.