CN113892481B

CN113892481B - Hydrogel for storage, transportation and bioprinting of cells or tissues

Info

Publication number: CN113892481B
Application number: CN202010577166.5A
Authority: CN
Inventors: 柯东旭; 杨熙; 蔡正伟
Original assignee: Suzhou Nuopu Regenerative Medicine Co ltd
Current assignee: Suzhou Nuopu Regenerative Medicine Co ltd
Priority date: 2020-06-22
Filing date: 2020-06-22
Publication date: 2022-09-20
Anticipated expiration: 2040-06-22
Also published as: CN113892481A

Abstract

The present invention relates to a hydrogel for storage, transport and bioprinting of cellular living tissue. Firstly, the application of adenosine for prolonging the storage time of cells or tissues in hydrogel can reach more than 20 days. When adenosine is used for storing and transporting hydrogel of cells or tissues, the storage time of the cells can be greatly prolonged, and the adenosine can be used for storing living cells for a long time; adenosine can maintain higher activity of cells in a hypoxic environment, thereby realizing bioprinting of large-volume tissues. The invention also provides a hydrogel for bioprinting cells or tissues, which comprises the following components dissolved in a cell culture medium: 0-30mM adenosine, 10-50mg/mL hydrogel polymer, 1-20% v/v serum, 0-5% v/v antibiotic, 10-50mg/mL gelatin, 0-5mg/mL hyaluronic acid, 0-20% v/v glycerol, and the amount of adenosine is not 0 mM.

Description

Hydrogel for storage, transportation and bioprinting of cells or tissues

Technical Field

The invention relates to the field of hydrogel, in particular to hydrogel for storage, transportation and bioprinting of cells or tissues.

Background

In the biological field, the use of cells is wide, including tissue engineering, vaccine production, cell therapy, in vitro test and the like, and in the application process, the storage and transportation of the cells are an important part, and how to maintain the activity of the cells in the storage and transportation and reduce the storage and transportation cost are important problems to be solved in the cell field.

The transportation method commonly used for the cells at present is the frozen transportation of cell sap or serum containing dimethyl sulfoxide (DMSO) at the temperature of minus 80 ℃ or under the condition of liquid nitrogen, and the transportation method has the advantage of being beneficial to the long-term storage of the cells. However, DMSO has a toxic effect, reacts with protein hydrophobic groups, causes protein denaturation, has vascular toxicity and hepatorenal toxicity, and is very unfavorable for the short-term storage of specific cells or primary cells and related gene tests. In addition, the ultra-low temperature storage method brings about an increase in cost. At present, the living tissue is usually transported by a method of storing the living tissue in a cell culture medium at 4 ℃, the shearing force of the shaking of cell sap on the tissue in the transportation process can influence the activity of the tissue, and in addition, the long-time transportation of the living tissue is very unfavorable because a common ice box can only keep the low temperature for about 12 to 24 hours.

There are documents disclosing encapsulation of cells with hydrogels for storage and transport of cells, the hydrogel components including sodium alginate, gelatin, dextran, PEG, albumin, polypeptides, etc. For example, patent CN104920339B discloses a method for preserving cells by using biocompatible particles: physically mixing hydrogel particles of pure biocompatible materials with cells, centrifuging and enriching, stacking the hydrogel particles to wrap the cells, and uniformly mixing the compatible particles with the cells in any appropriate proportion; the technical effect is that the preservation method is a physical preservation method, and the vitality of the cells can be maintained under the severe transportation and preservation conditions. However, the hydrogel in this patent stores cancer cells, and the preservation effect of normal cells is not demonstrated. Patent EP2688397B1 discloses a composition comprising a hydrogel in which a cell population is encapsulated or entrapped, wherein the hydrogel is a 1.1-1.3% strontium alginate hydrogel, wherein the hydrogel is in the form of a thin layer or disc, wherein the composition is packaged in a form suitable for transport to a remote location; the technical effects are that the cells are fixed in the strontium alginate gel, the viability of the cells is improved, the pore size of the gel is controllable, the cells are easily released from the gel, and the survival rate is further improved. The hydrogel of this patent still has a short time to preserve cells and can be stored for only 7 days.

In addition, the cells have great application value in the aspect of human tissue repair, and tissues and organs can be reconstructed by co-culturing tissue engineering scaffolds and the cells or biologically printing hydrogel containing the cells. However, the bioprinting technology has a bottleneck for the printing of large-volume human tissues, and since the permeation range of oxygen is only 200 μm, the center of the large-volume human tissues subjected to bioprinting is anoxic and necrotized, and the bioprinting of implantable complex organs cannot be realized. Therefore, the method can improve the hypoxia tolerance of cells in hydrogel and ensure the activity of cells, and is a technical problem to be solved urgently. The current hydrogel for cell preservation cannot solve the storage problem of cells in a hypoxic environment, and is not suitable for directly being used for bioprinting large-volume tissues.

In view of the above, the present invention provides a hydrogel for storing, transporting and bioprinting cells or tissues, which is capable of solving the technical problems of long-term storage and transportation of cells or tissues at room temperature and necrosis of cells in large-volume tissues.

Disclosure of Invention

The present hydrogel is used for storing cells or tissues, and the activity of the cells is rapidly reduced and the cells die in a short time under the normal temperature or hypoxia environment, but the inventor finds that when adenosine is used for storing and transporting the hydrogel of the cells or tissues, the storage time of the cells can be greatly prolonged, and the storage activity of the cells can be improved, and particularly the hydrogel is used in the normal temperature and hypoxia environment.

The first object of the present invention is to provide the use of adenosine in the storage of cell or tissue hydrogels for prolonging the storage time of cells, improving the storage activity of cells, in particular the storage of cells in hydrogels under normal temperature and/or hypoxic conditions.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

use of adenosine for prolonging the storage time of cells or tissues in a hydrogel, the prolonged storage time being 11 days or more, preferably 17 days or more, more preferably 20 days or more.

Use of adenosine for prolonging the storage time of cells or tissues in a hydrogel, the storage being able to be performed at ambient temperature, which is 0-37 ℃, preferably 17-27 ℃, as adenosine is able to reduce cellular metabolism.

Meanwhile, after being stored for a longer time, the cells still have higher activity; further, the cell activity is greater than 4, preferably greater than 6, at 11 days of storage; the cell activity refers to the ratio of the MTT test OD value to the MTT test OD value of the cell prior to storage.

Use of adenosine to prolong the storage time of cells or tissues in a hydrogel, said storage being under hypoxic conditions, which means that the oxygen content may be below 5%, or below 2%, or below 1%, such as 0.1%. The hydrogel containing adenosine can store cells in a hypoxic environment, and after the hydrogel is used for bioprinting a large-volume tissue, the inside of the large-volume tissue is in the hypoxic environment, and the cells can still keep certain activity for a long time, so that the tissue repair effect is improved.

Meanwhile, the cells still have higher activity after being stored for a longer time under the hypoxia condition; further, the cell activity is greater than 1, preferably greater than 1.5, and more preferably greater than 1.7 at 11 days of storage; or a cell activity of greater than 1, preferably greater than 1.3, more preferably greater than 1.5, when stored for 17 days; or a cell activity greater than 0.4, preferably greater than 0.5, when stored for 20 days; the cell activity refers to the ratio of the MTT test OD value to the MTT test OD value of the cell prior to storage.

The second object of the present invention is to provide a hydrogel for storage and transportation of cells or tissues, which can store cells for a long time, maintain the higher activity of the cells, and the storage conditions can be a normal temperature and/or a hypoxic environment.

The hydrogel for storing and transporting the cells or the tissues comprises the following components dissolved in a cell culture medium: 0-30mM adenosine, 10-50mg/mL hydrogel polymer, and the dosage of adenosine is not 0 mM;

further, the following components are dissolved in the cell culture medium: 10-30mM adenosine, 20-50mg/mL hydrogel polymer.

Further, the hydrogel for storing and transporting cells or tissues also comprises the following components dissolved in a cell culture medium: 1-20% v/v serum, 0-5% v/v antibiotic and a suitable amount of cross-linking agent.

Further, the hydrogel further comprises 4500mg/L glucose, which provides sufficient energy to the cells.

The hydrogel polymer can be one or more of sodium alginate, gelatin, hyaluronic acid, polyethylene glycol, fibrin and the like;

the cross-linking agent can be selected from calcium chloride or thrombin;

the cell culture medium can be one or more selected from DMEM, RPMI 1640, MEM, DMEM/F12 and the like;

the antibiotic may be selected to be streptomycin.

In one exemplary embodiment, the hydrogel comprises the following components dissolved in DMEM medium: 30mM/mL adenosine, 30mg/mL fibrin, 20UI/mL thrombin, 10% v/v serum, 1% v/v penicillin.

The third purpose of the invention is to provide the hydrogel for bioprinting the cells or the tissues, and the bioprinting tissue for wrapping the cells or the tissues is obtained by bioprinting.

When the hydrogel is used for bioprinting, it further comprises glycerol, gelatin and hyaluronic acid.

The hydrogel for bioprinting cells or tissues comprises the following components dissolved in a cell culture medium: 0-30mM adenosine, 10-50mg/mL hydrogel polymer, 1-20% v/v serum, 0-5% v/v antibiotic, 10-50mg/mL gelatin, 0-5mg/mL hyaluronic acid, 0-20% v/v glycerol and a proper amount of cross-linking agent.

In one exemplary embodiment, the hydrogel for bioprinting cells or tissues comprises the following components dissolved in a cell culture medium: 30mM adenosine, 30mg/mL fibrin, 10% v/v serum, 20UI/mL thrombin, 1% v/v antibiotic, 30mg/mL gelatin, 3mg/mL hyaluronic acid, 10% v/v glycerol.

In addition, the first object of the present invention, the use of adenosine for prolonging the storage time of cells or tissues in a hydrogel, wherein said hydrogel may be selected from the hydrogels provided for the second and third objects of the present invention.

The preparation method of the hydrogel for storing and transporting the cells or tissues or the hydrogel for bioprinting the cells or tissues comprises the following steps:

the components are uniformly mixed according to a proportion to obtain a mixed solution, cells or tissues are mixed with the mixed solution to obtain a hydrogel solution containing the cells or the tissues, a cross-linking agent is added into the hydrogel solution to complete the solidification of the hydrogel, then the cells or the tissues are stored and/or transported, or the hydrogel solution is subjected to biological printing, the cross-linking agent is added in the printing process or after the printing process is finished to obtain the printed tissues, and then the tissue culture is carried out.

A typical preparation method comprises the following steps: DMEM, water, adenosine, glucose, glycerol, hyaluronic acid and streptomycin are mixed for 4-16 hours by a rotary mixing device according to a certain proportion to obtain a first mixed solution; adding quantitative gelatin and fibrin into the first solution, and continuously mixing for 1-5 hours to obtain a second solution; adding cells or tissues to obtain a hydrogel solution; adding thrombin into the hydrogel solution to crosslink the hydrogel for storing and transporting cells or tissues, or performing bioprinting on the hydrogel solution, adding thrombin during bioprinting, solidifying the printed tissues, and then performing tissue culture.

Compared with the prior art, the invention has the beneficial effects that:

1. when the adenosine is used for storing and transporting the hydrogel of cells or tissues, the storage time of the cells can be greatly prolonged, the prolonged storage time can reach more than 20 days, the storage activity of the cells can be improved, and the activity of the stored cells can reach more than 6 on day 11.

2. The adenosine-containing hydrogel can reduce the metabolic activity of cells, so that the cells can be stored and transported for a long time at normal temperature (for example, 17-27 ℃) and the storage and transportation cost of the cells is reduced.

3. The hydrogel containing adenosine can prolong the storage time of cells in a hypoxic environment and keep higher activity of the cells in the hypoxic environment, so that the biological printing of large-volume tissues is realized, and the necrosis of the cells in the hypoxic environment in the center of the large-volume tissues is avoided; under extreme hypoxic conditions of 0.1%, cell activity still reached 1.35 at day 17.

4. Fibrin in the hydrogel system has good biocompatibility; the addition of streptomycin can avoid accidental infection; the application of gelatin, glycerol and hyaluronic acid is beneficial to the flowing and biological printing of hydrogel; the use of glucose provides the cells with sufficient energy.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 shows the cell activity of mouse C2C12 cells stored in the hydrogels of example 1 and comparative example 1 in different temperature cultures;

FIG. 2 shows the cell viability of mouse C2C12 cells stored at 37 ℃ in the hydrogels of example 1 and comparative example 1, cultured in an extreme condition of 0.1% oxygen;

FIG. 3 is a drawing of a hydrogel bioprint design, photo imaging and photograph of example 2 and comparative example 2;

figure 4 is a graph of hematoxylin and eosin staining of hydrogel bioprinted tissues of example 2 and comparative example 2 two weeks after subcutaneous implantation in mice.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1

A hydrogel for storage and transportation of cells or tissues, which comprises the following components dissolved in a DMEM medium: 30mM adenosine, 30mg/mL fibrin, 20UI/mL thrombin, 10% v/v serum, 1% v/v streptomycin.

Mixing DMEM, water, adenosine, glucose and streptomycin at a certain ratio by a rotary mixing device at 37 ℃ for 12 hours to obtain a first mixed solution; adding fibrin into the first solution and continuously mixing for 2 hours to obtain a second solution; adding mouse C2C12 cells to obtain a hydrogel solution; thrombin was added to the hydrogel solution to crosslink the hydrogel for storage and transportation of mouse C2C12 cells.

Example 2

A hydrogel for bioprinting cells or tissues, comprising the following components dissolved in DMEM medium: 30mM adenosine, 30mg/mL fibrin, 10% v/v serum, 20UI/mL thrombin, 1% v/v streptomycin, 30mg/mL gelatin, 3mg/mL hyaluronic acid, 10% v/v glycerol.

Mixing DMEM, water, adenosine, glycerol, hyaluronic acid and streptomycin at a ratio by a rotary mixing device at 37 ℃ for 12 hours to obtain a first mixed solution; adding gelatin and fibrin into the first solution, and continuously mixing for 2 hours to obtain a second solution; adding mouse C2C12 cells to obtain a hydrogel solution; and (3) carrying out biological printing on the hydrogel solution, adding thrombin in the biological printing process, solidifying the printed tissue, and then carrying out tissue culture.

Example 3

A hydrogel for storage and transportation of cells or tissues, comprising the following components dissolved in a DMEM medium: 10mM adenosine, 20mg/mL sodium alginate, 50mg/mL calcium chloride, 10% v/v serum, 1% v/v streptomycin.

Mixing DMEM, water, adenosine, glucose and streptomycin at a certain ratio by a rotary mixing device at 37 ℃ for 12 hours to obtain a first mixed solution; adding sodium alginate into the first solution and continuously mixing for 2 hours to obtain a second solution; adding mouse C2C12 cells to obtain a hydrogel solution; calcium chloride is added into the hydrogel solution to crosslink the hydrogel for storing and transporting cells or tissues.

Example 4

A hydrogel for bioprinting cells or tissues, comprising the following components dissolved in DMEM medium: 10mM adenosine, 20mg/mL sodium alginate, 10% v/v serum, 50mg/mL calcium chloride, 1% v/v streptomycin, 30mg/mL gelatin, 3mg/mL hyaluronic acid, 10% v/v glycerol.

Mixing DMEM, water, adenosine, glycerol, hyaluronic acid and streptomycin at a certain ratio by a rotary mixing device at 37 ℃ for 12 hours to obtain a first mixed solution; adding gelatin and sodium alginate into the first solution, and continuously mixing for 2 hours to obtain a second solution; adding mouse C2C12 cells to obtain a hydrogel solution; and (3) carrying out biological printing on the hydrogel solution, adding calcium chloride in the biological printing process, solidifying the printed tissue, and then carrying out tissue culture.

Comparative example 1

The difference from the example 1 is that: adenosine is not present.

Comparative example 2

The difference from the embodiment 2 is that: adenosine is not present.

The experimental results are as follows:

(1) mouse C2C12 cells were stored in the hydrogels of example 1 and comparative example 1, cell activity at different temperature cultures:

mouse C2C12 cells were stored in the hydrogels of example 1 and comparative example 1, cultured in incubators at 7 deg.C, 17 deg.C, 27 deg.C and 37 deg.C, respectively, and subjected to MTT cell viability test on

day

0, 7 and 11, as shown in FIG. 1, wherein comparative example 1 is a control group and example 1 is an experimental group. Wherein day 0 is the MTT assay of the cells prior to storage and the OD is 0.28.

As can be seen from FIG. 1, the mouse C2C12 cells stored in the hydrogel of example 1 showed better effect at 17 deg.C, 27 deg.C and 37 deg.C, and on day 11, the cells still maintained higher activity with OD values of 1.86, 1.70 and 1.28, which were calculated to be about 6.64, 6.07 and 4.57, respectively, where the cell activity refers to the ratio of the MTT test OD value to the MTT test OD value of the cells before storage.

As can be seen by comparing example 1 with comparative example 1, the cell storage activities were similar for both cells by day 7, e.g., the OD value of example 1 was 2.30 at 17 ℃ and 2.00 for comparative example 1, which were calculated to be about 8.21 and 7.14, respectively; however, on day 11, the cell activity of comparative example 1, to which adenosine was not added, was drastically decreased, and the OD at 17 ℃ was 0.85, and the cell activity was about 3.04, whereas example 1, to which adenosine was added, maintained a high cell activity, and the OD at 17 ℃ was 1.86, and the cell activity was about 6.64. It can thus be demonstrated that adenosine is able to greatly prolong the storage time of cells or tissues in hydrogels. In addition, the example 1 and the comparative example 1 have poor effect of storing cells or tissues at a temperature of 7 ℃, and the activity of the cells is continuously reduced from the 7 th to the 11 th days, which is not good for storing the cells, but at the same time, the example 1 has better effect than the comparative example 1.

(2) Mouse C2C12 cells were stored in example 1 and comparative example 1 hydrogels at 37 ℃ for cell activity in extreme cases of 0.1% oxygen culture:

mouse C2C12 cells were stored in the example 1 and comparative example 1 hydrogels at 37 deg.C, cultured at 0.1% oxygen extremes, and tested for cell viability on

days

7, 11, 17, and 20, as shown in FIG. 2. Wherein comparative example 1 is a control group and example 1 is an experimental group. Wherein day 0 is the MTT assay of the cells prior to storage and the OD is 0.20.

As can be seen from fig. 2, the mouse C2C12 cells stored in the hydrogel of comparative example 1 had higher cell activity at day 7, OD value of 0.53 and cell activity of 2.65, but rapidly necrosed after 7 days, and very low cell activity after 11 days, OD value of 0.037 and cell activity of about 0.185. Whereas mouse C2C12 cells were stored in the hydrogel of example 1, although the cell activity at day 7 was slightly lower than in comparative example 1, OD value was 0.35, cell activity was 1.75; after day 11, however, the cell viability was much higher than in comparative example 1, with an OD of 0.34, and the cell viability was approximately 1.7: the cell activity was about 1.35 at day 17, and was maintained at a value of 0.089 OD and about 0.45 at day 20. It was thus demonstrated that adenosine can greatly prolong the storage time of cells or tissues in hydrogels, even under extreme conditions of 0.1% oxygen content, for more than 20 days.

(3) Hematoxylin eosin staining of hydrogel bioprinted tissues of example 2 and comparative example 2 two weeks after subcutaneous implantation in mice:

first, a bioprinted design was performed, and then the hydrogels of example 2 and comparative example 2 were prepared into bioprinted tissues, the design drawings, optical imaging drawings, and photographs of which are shown in fig. 3 (a) (b) (c) in this order.

The hydrogel bioprinted tissues of example 2 and comparative example 2 were implanted subcutaneously in mice for two weeks and stained with hematoxylin and eosin, wherein comparative example 2 was a control group and example 2 was an experimental group, and the results are shown in fig. 4. In fig. 4, the light color in the center of the control group image is unstained tissue, indicating that apoptosis and tissue exfoliation occur in the center of the tissue, while the center of the experimental group image is stained, indicating that the center of the tissue grows well. Therefore, the hydrogel organism disclosed by the invention is used for printing large-volume tissues, and the technical problem of oxygen-deficient necrosis of central cells of the tissues is solved.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of adenosine in prolonging the storage time of cells or tissues in hydrogel is characterized in that the prolonged storage time is more than 11 days, the storage is carried out under the condition of normal temperature, and the normal temperature is 17-27 ℃.

2. Use according to claim 1, characterized in that the storage time after prolonged is more than 17 days.

3. Use according to claim 1 or 2, wherein the cell activity is greater than 4 at 11 days of storage; the cell activity refers to the ratio of the MTT assay OD value to the MTT assay OD value of the cell prior to storage.

4. Use according to claim 1, characterized in that said storage is carried out under hypoxic conditions, said hypoxic meaning an oxygen content lower than 5%; when the storage time is 11 days, the cell activity is more than 1; or the cell activity is more than 1 when the storage time is 17 days; or when the storage time is 20 days, the cell activity is more than 0.4; the cell activity refers to the ratio of the MTT test OD value to the MTT test OD value of the cell prior to storage.

5. The hydrogel according to claim 1 for storage and transportation of cells or tissues, comprising the following components dissolved in a cell culture medium: 0-30mM adenosine, 10-50mg/mL hydrogel polymer, and adenosine is not used in an amount of 0 mM.

6. The hydrogel according to claim 5, further comprising the following components dissolved in a cell culture medium: 1-20% v/v serum, 0-5% v/v antibiotic.

7. The hydrogel according to claim 6, wherein the hydrogel polymer is selected from one or more of sodium alginate, gelatin, hyaluronic acid, polyethylene glycol and fibrin; the cell culture medium is selected from one or more of DMEM, RPMI 1640, MEM and DMEM/F12; the antibiotic is selected to be streptomycin.

8. The hydrogel according to claim 5, which comprises the following components dissolved in DMEM medium: 30mM/mL adenosine, 30mg/mL fibrin, 20UI/mL thrombin, 10% v/v serum, 1% v/v penicillin.

9. A hydrogel according to claim 1 for bioprinting of cells or tissue, comprising the following components dissolved in a cell culture medium: 0-30mM adenosine, 10-50mg/mL hydrogel polymer, 1-20% v/v serum, 0-5% v/v antibiotic, 10-50mg/mL gelatin, 0-5mg/mL hyaluronic acid, 0-20% v/v glycerol, and the amount of adenosine is not 0 mM.

10. The hydrogel according to claim 9, which comprises the following components dissolved in a cell culture medium: 30mM adenosine, 30mg/mL fibrin, 10% v/v serum, 20UI/mL thrombin, 1% v/v streptomycin, 30mg/mL gelatin, 3mg/mL hyaluronic acid, 10% v/v glycerol.