CN114375944A - A kind of cryopreservation solution and cryopreservation method for bone tissue and osteochondral tissue - Google Patents

Abstract

The invention belongs to the technical field of tissue cryopreservation, and particularly relates to a cryopreservation liquid and a cryopreservation method for bone tissues and bone soft tissues. The invention provides a novel cryopreservation liquid for bone tissues and bone soft tissues and also provides a method for performing cryopreservation on the bone tissues and the bone soft tissues by using the cryopreservation liquid. The cryopreservation liquid is particularly suitable for cryopreservation of bone tissues and bone soft tissues, can effectively reduce the toxicity of the low-temperature protective agent in the cryopreservation liquid to cells, improve the survival rate of the cells, and reduce the damage to the cells and the tissues, thereby being beneficial to prolonging the preservation time of the bone tissues and the bone soft tissues in vitro and having good application prospect.

Description

A kind of cryopreservation solution and cryopreservation method for bone tissue and osteochondral tissue

technical field

The invention belongs to the technical field of tissue cryopreservation, in particular to a cryopreservation solution and cryopreservation method for bone tissue and soft bone tissue.

Background technique

In clinical practice, bone defects caused by trauma, infection, bone tumor resection and other reasons are one of the difficult problems in orthopaedic treatment, and bone transplantation is the preferred treatment method. Although autologous bone grafting is the "gold standard" for bone defect repair, the increased trauma caused by bone harvesting and the limited amount of bone harvested limit its clinical application. Allogeneic bone comes from a wide range of sources, produces little rejection, and does not cause secondary injury, which solves the problem of insufficient tissue sources for autologous bone grafting, so it is widely used in clinical practice. How to preserve the allogeneic bone and osteochondral tissue from the donor for a long time in vitro with good cell activity has become the focus of research by many scholars.

Existing cryopreservation methods mainly include programmed method and vitrification method. The programmed method takes a long time and easily produces ice crystals in the cells. The vitrification method can avoid the generation of intracellular ice crystals and is easy to operate, which has become a hot spot in cryopreservation research in recent years. However, there are many factors affecting the cryopreservation effect of the vitrification method, and a unified and standardized method has not yet been formed. Especially the cryopreservation solution, which plays a crucial role in the cryopreservation effect. Cryoprotectants are an important part of cryopreservation, which can protect cells from being damaged by low temperature.

However, cryoprotectants are generally toxic substances. Therefore, how to design the formula of cryopreservation solution, and how to set the method for cryopreservation of cells, so as to reduce the toxicity of cryoprotectant to cells as much as possible are important research topics in the field of cryopreservation of cells. The Chinese invention patent application "CN201910853381.0 An immune cell vitrification cryopreservation protection solution and a method for cryopreservation of immune cells" proposes a cryopreservation solution and method for immune cells. The use of the protective agent dimethyl sulfoxide or ethylene glycol reduces the toxicity of the cryopreservation solution and improves the cell viability.

For the cryopreservation of bone tissue and soft tissue, the existing cryopreservation solutions and cryopreservation methods have problems of excessive toxicity and low cell viability. Therefore, it is necessary to develop new cryopreservation solutions for bone tissue and soft tissue Freezing method.

SUMMARY OF THE INVENTION

Aiming at the defects of the prior art, the present invention provides a cryopreservation solution and a cryopreservation method for bone tissue and soft tissue, the purpose of which is to reduce the toxicity of the cryopreservation solution by optimizing the formula and cryopreservation method of the cryopreservation solution. , which provides the viability of cryopreserved cells.

A freezing solution for bone tissue and/or osteochondral tissue, each 1000ml of the freezing solution is composed of the following components:

KOH 80-120mmol,

HCl 40-80mmol,

NaHCO ₃ 10-50mmol,

NaH ₂ PO ₄ 0.8-1.2mmol,

MgSO ₄ 0.8-1.2 mmol,

CaCl ₂ 0.8-1.2mmol,

Glucose 2-8mmol,

TES 80-120mmol,

1,2 Propylene Glycol 80-120ml,

99.9% DMSO 80-120ml,

The remainder is water.

Preferably, each 1000ml of the freezing solution consists of the following components:

KOH 100mmol,

HCl 60mmol,

NaHCO3 30mmol,

NaH2PO4 1mmol,

MgSO4 1mmol,

CaCl2 1mmol,

Glucose 5mmol,

TES 100mmol,

1,2 Propylene Glycol 100ml,

99.9% dimethyl sulfoxide 100ml,

The balance is deionized water.

Preferably, the pH of the cryopreservation solution is 7.3-7.5.

The present invention also provides the use of the above cryopreservation solution in cryopreserving bone tissue and/or soft bone tissue.

The present invention also provides a cryopreservation method for bone tissue and/or osteochondral tissue, comprising the following steps:

Step 1, prepare the above freezing solution;

Step 2, place the tissue to be frozen in the freezing solution at 0-4°C for 1-3 hours;

Step 3: Gradually cool down the tissue and cryopreservation solution to be frozen to -70--90°C, place for 8-12 hours, and then store in liquid nitrogen.

Preferably, in step 2, before putting the tissue to be frozen into the cryopreservation solution, it is washed 3-5 times with a phosphate buffer.

Preferably, in step 3, the stepwise cooling rate is -0.1～-0.3°C/min.

In the present invention, "TES" is the compound 2-[[tris(hydroxymethyl)methyl]amino]ethanesulfonic acid.

The invention optimizes the composition of the cryopreservation solution for bone tissue and bone soft tissue, uses only dimethyl sulfoxide as a cryoprotectant, and minimizes the damage to cells caused by the cryoprotectant. In addition, the invention also improves the cryopreservation method, does not perform rapid heating or cooling, and utilizes the characteristic that the toxicity of the cryoprotectant dimethyl sulfoxide decreases with the decrease of temperature, when preserving bone tissue or bone cartilage tissue. , gradually cool down, and finally make enough dimethyl sulfoxide in the tissue cells at low temperature to achieve vitrification. This further reduces damage to cells by DMSO. It can be seen that the present invention not only provides a new cryopreservation solution for the cryopreservation of bone tissue and soft bone tissue, but also minimizes the damage to cells caused by the cryopreservation solution, which can effectively improve the cryopreservation of bone tissue and soft bone tissue. It can prolong the storage time of bone tissue and soft bone tissue in vitro, so as to facilitate the establishment of bone tissue and bone soft tissue bank, and expand the clinical application of allogeneic bone transplantation in repairing bone defects. Therefore, the present invention has a good application prospect.

Obviously, according to the above-mentioned content of the present invention, according to the common technical knowledge and conventional means in the field, without departing from the above-mentioned basic technical idea of the present invention, other various forms of modification, replacement or change can also be made.

The above content of the present invention will be further described in detail below through the specific implementation in the form of examples. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention.

Description of drawings

Fig. 1 is the experimental result of the survival rate of cryopreserved cells in Experimental Example 1;

Fig. 2 is the HE staining image of the bone tissue and cartilage tissue frozen according to the cryopreservation solution and cryopreservation method of Example 3 in Experimental Example 1;

FIG. 3 is an HE staining image of the bone tissue and cartilage tissue cryopreserved according to the cryopreservation solution and cryopreservation method of Comparative Example 1 in Experimental Example 1. FIG.

Detailed ways

In the following examples and experimental examples, all reagents and materials not specifically described are commercially available.

Example 1

Bone tissue and bone soft tissue cryopreservation solution of the present invention, each 1000ml contains KOH 80-120mmol, HCl 40mmol, NaHCO ₃ 10mmol, NaH ₂ PO ₄ 0.8mmol, MgSO ₄ 0.8mmol, CaCl ₂ 0.8mmol, glucose 2mmol, TES80mmol, 1, 2 80 ml of propylene glycol, 80 ml of 99.9% dimethyl sulfoxide, and the balance is deionized water. The pH of this bone and soft tissue cryopreservation solution is 7.3-7.5. Aliquot, seal, and store at 0-4°C.

Example 2

The bone tissue and bone and soft tissue cryopreservation solution of the present invention, the bone tissue and bone and soft tissue cryopreservation solution of the present invention, each 1000ml contains KOH 120mmol, HCl 80mmol, NaHCO ₃ 50mmol, NaH ₂ PO ₄ 1.2mmol, MgSO ₄ 1.2mmol, CaCl ₂ 1.2 mmol, 8 mmol of glucose, 120 mmol of TES, 120 ml of 1,2 propylene glycol, 120 ml of 99.9% dimethyl sulfoxide, and the balance is deionized water. The pH of this bone and soft tissue cryopreservation solution is 7.3-7.5. Aliquot, seal, and store at 0-4°C.

Example 3

Bone tissue and soft tissue cryopreservation solution of the present invention, each 1000ml contains KOH 100mmol, HCl 60mmol, NaHCO ₃ 30mmol, NaH ₂ PO ₄ 1mmol, MgSO ₄ 1mmol, CaCl ₂ 1 mmol, glucose 5mmol, 2-[[tris(hydroxymethyl) base) methyl] amino] ethanesulfonic acid 100 mmol, 100 ml of 1,2 propylene glycol, 100 ml of 99.9% dimethyl sulfoxide, the balance is deionized water, the pH value of the bone tissue and soft tissue cryopreservation solution is 7.3-7.5. Aliquot, seal, and store at 0-4°C.

The method for freezing bone tissue and osteochondral tissue using the cryopreservation solution of the present embodiment is as follows:

Step 1, prepare the above freezing solution;

Step 2, the tissue to be cryopreserved is first washed 3-5 times with phosphate buffer, and then incubated in the cryopreservation solution at 4°C for 2 hours;

Step 3, the tissue and cryopreservation solution to be frozen are gradually cooled and frozen to -80°C at a speed of -0.3°C/min, placed for 12 hours, and then stored in liquid nitrogen.

The technical effects of the present invention will be further described below through experiments.

Experimental example 1

1. Experimental method

The osteochondral tissue was cryopreserved by using the cryopreservation solution of Example 3 (with 10% dimethyl sulfoxide cryopreservation solution as the control group) and the cryopreservation method, and the chondrocyte survival rate of the cryopreserved osteochondral tissue was investigated and collected HE stained images.

The specific method to obtain the chondrocyte survival rate is as follows: directly soak the cryopreservation tube in a 37°C water bath for rewarming, take out the osteochondral block, make sections, stain, and then observe the chondrocytes under a fluorescence microscope. The chondrocyte survival rate was the number of green-stained cells/(the number of green-stained cells + the number of orange-stained cells).

2. Experimental results

The survival rate of cryopreserved cells is shown in Figure 1. It can be seen from the figure that the survival rate of cells in the control group was 84% at week 2, 79% at week 4, and 72% at week 6. The invention group (using the cryopreservation solution and cryopreservation method of Example 3) had a cell survival rate of 89% in the second week, 84% in the fourth week, and 79% in the sixth week. It can be seen that the present invention can effectively improve the cell survival rate.

Figure 2 shows the HE staining images of bone tissue and cartilage tissue frozen according to the cryopreservation solution and cryopreservation method of Example 3, and Figure 3 shows the HE staining images of the control group. As can be seen from the figure, the articular surface of the control group was significantly damaged, the chondrocytes were arranged disorderly and aggregated more, the cartilage damage was obvious, and the subchondral trabecular structure was reduced, slender and disordered. The number of articular subchondral trabeculae was significantly reduced and resulted in microstructural damage. On the other hand, in the experimental group of Example 3, the articular surface was relatively complete, the chondrocyte morphology was relatively regular, and the subchondral bone trabecular structure was relatively neatly arranged. It can be seen that the cryopreservation solution and cryopreservation method of the present invention cause less damage to bone tissue and soft tissue, and are more conducive to long-term preservation of them.

It can be seen from the above examples and experimental examples that the present invention provides a new cryopreservation solution and a cryopreservation method. The invention is particularly suitable for cryopreservation of bone tissue and soft tissue, and can effectively reduce the cryoprotection in the cryopreservation solution. Toxicity of agents to cells, increase cell viability, and reduce damage to cells and tissues. Thereby, it is beneficial to prolong the preservation time of the bone tissue and the bone soft tissue in vitro, so as to facilitate the establishment of the bone tissue and the bone soft tissue bank, and to expand the clinical application of allogeneic bone transplantation in repairing bone defects. Therefore, the present invention has a good application prospect.

Claims (7)

1. A cryopreservation liquid for bone tissue and/or soft bone tissue, wherein each 1000ml of the cryopreservation liquid comprises the following components:

KOH 80-120mmol、

HCl 40-80mmol、

NaHCO₃ 10-50mmol、

NaH₂PO₄ 0.8-1.2mmol、

MgSO₄ 0.8-1.2mmol、

CaCl₂ 0.8-1.2mmol、

2-8mmol of glucose,

TES 80-120mmol、

80-120ml of 1, 2-propylene glycol,

80-120ml of 99.9 percent dimethyl sulfoxide,

the balance being water.

2. The cryopreservation liquid of claim 1, wherein each 1000ml of the cryopreservation liquid is composed of:

KOH 100mmol、

HCl 60mmol、

NaHCO3 30mmol、

NaH2PO4 1mmol、

MgSO4 1mmol、

CaCl2 1mmol、

5mmol of glucose,

TES 100mmol、

100ml of 1, 2-propanediol,

100ml of 99.9% dimethyl sulfoxide was added,

the balance being deionized water.

3. The frozen stock solution according to claim 1 or 2, wherein: the pH value of the frozen stock solution is 7.3-7.5.

4. Use of the cryopreservation solution of any one of claims 1 to 3 for the cryopreservation of bone tissue and/or bone soft tissue.

5. A method for cryopreserving bone tissue and/or soft bone tissue, comprising the steps of:

step 1, preparing a cryopreservation solution according to any one of claims 1 to 3;

step 2, incubating the tissue to be cryopreserved in a cryopreservation solution at 0-4 ℃ for 1-3 hours;

and 3, gradually cooling and freezing the tissues to be frozen and the frozen stock solution to-70 to-90 ℃, standing for 8 to 12 hours, and then storing in liquid nitrogen.

6. The cryopreservation method of claim 5, wherein: in step 2, before the tissue to be frozen is put into the freezing medium, phosphate buffer solution is adopted to wash the tissue for 3 to 5 times.

7. The cryopreservation method of claim 5, wherein: in the step 3, the gradual cooling speed is-0.1 to-0.3 ℃/min.

Images