CN113767895B - Composite hair follicle tissue preserving fluid, preparation method thereof and preserving method for maintaining hair follicle in vitro activity - Google Patents

Abstract

The invention relates to a composite preservation solution for hair follicle tissue, a preparation method thereof and a preservation method for maintaining the in vitro activity of hair follicles, belonging to the technical field of cell cryopreservation. The preservation method for maintaining the in vitro activity of hair follicles includes the following steps: 1. Take the composite preservation solution of hair follicle tissue, pre-cooled at 4°C; take the isolated unit hair follicles, rinse with Ringer's solution to remove blood clots and impurities; The hair follicles are placed in the cold hair follicle tissue composite preservation solution, fully immersed, and the storage temperature should be maintained at 4 °C; 2. Remove the hair follicle tissue composite preservation solution, rinse the unit hair follicle with Ringer's solution, and remove the residual preservation solution. in vivo. The hair follicle tissue composite preservation solution is composed of a basic preservation solution and an auxiliary preservation solution, and the basic preservation solution is composed of a solute, a solvent and a pH value adjuster. The invention has the following advantages: no risky suspicious components, the preserved hair follicles can be directly used for clinical transplantation treatment after being washed with simple physiological saline, which is safe and effective, and has a good clinical application prospect.

Description

A kind of hair follicle tissue composite preservation solution, its preparation method and maintaining hair follicle in vitro viability Sexual preservation method

technical field

The invention belongs to the technical field of cell cryopreservation, and in particular relates to a composite preservation solution for hair follicle tissue, a preparation method thereof, and a preservation method for maintaining the in vitro activity of hair follicles.

Background technique

Androgenic alopecia (AGA) is one of the most common skin diseases in the world, afflicting more than 100 million adult men in my country. Although AGA does not affect physical health, the appearance of baldness has a great psychological impact on patients, and even some patients have social barriers. As an important means of clinical treatment of AGA, surgical hair transplantation is mainly aimed at severe and late irreversible AGA hair loss. With the development of technology, it is also suitable for mild to moderate patients. Currently, the main surgical treatment option involves drilling a healthy unit of hair follicles in the posterior occipital region, followed by perforation and transplantation into the bald area. However, autologous hair transplantation is an extremely time-consuming procedure, and the operation time increases exponentially as the number of transplanted follicles increases. For small area unit hair follicle transplantation surgery (about 2000 units), it usually takes 3 hours to obtain the hair follicle and 3 hours for the implantation process. For large-area follicular unit transplantation (about 4,000 units), the total operation time is close to 8-10 hours. In the current surgical process, the extracted hair follicles inevitably need to undergo a long period of in vitro environment before they can be transplanted into the body again. The huge environmental difference between in vivo and in vitro is a huge test for the survival of hair follicle cells. How to maintain the best viability of isolated hair follicles is one of the most critical determinants of the survival rate of transplanted hair follicles in hair transplantation.

There are many factors affecting the activity of isolated hair follicles, which can be mainly divided into two aspects: hair follicle preservation temperature and hair follicle preservation solution. For the storage temperature of hair follicles, current research has confirmed that 4°C is the best storage temperature for hair follicles in vitro, in order to reduce the metabolism of hair follicle cells and ensure the survival rate of hair follicles after transplantation. However, for hair follicle preservation solution, the most common in vitro preservation solution for hair follicle surgery is Ringer's solution (compound sodium chloride injection), which contains sodium ion, potassium ion, calcium ion, Magnesium, chloride and lactate ions. However, its high sodium properties may lead to cell edema at low temperature, and because of its lack of protective components, it is not conducive to long-term in vitro preservation of hair follicles, so it is not the best choice for in vitro preservation.

SUMMARY OF THE INVENTION

Based on the above reasons, a composite preservation solution of hair follicle tissue suitable for hair follicle transplantation and its preservation scheme have been developed, which have great application potential and commercial value for improving the effect of surgical treatment. At present, there is no commercial preservation solution reagent suitable for in vitro preservation of hair follicles on the market, which fails to meet the needs of future clinical use.

The first object of the present invention is to disclose a composite preservation solution for hair follicle tissue.

The second object of the present invention is to disclose a preparation method of a composite preservation solution for hair follicle tissue

The third object of the present invention is to disclose a preservation method for maintaining the viability of hair follicles in vitro.

The purpose of this invention is to realize through the following technical solutions:

A hair follicle tissue composite preservation solution, wherein: the hair follicle tissue composite preservation solution is composed of a basic preservation solution and an auxiliary preservation solution, wherein the basic preservation solution is composed of a solute, a solvent and a pH value regulator;

The pH adjuster is sodium hydroxide/hydrochloric acid, which is used to adjust the pH of the hair follicle tissue composite preservation solution to 7.4;

The solvent is medical sterile water; a part of the solvent is used to dissolve the solute to form a solution, and the volume of the solution is 1/2 of the volume of the composite preservation solution of the hair follicle tissue; the other part of the solvent is used to add the auxiliary preservation solution to the volume of the solution to the volume of hair follicle tissue composite preservation solution;

In terms of the volume of hair follicle tissue composite preservation solution, the composition of the solute is:

Sodium chloride 5-30 mmol/L

Potassium chloride 10-20 mmol/L

Magnesium chloride hexahydrate 1-10 mmol/L

Lacturonic acid 25-75 mmol/L

Trehalose 5-20 mmol/L

Mannitol 10-50 mmol/L

Potassium dihydrogen phosphate 5-50 mmol/L

In terms of the volume of hair follicle tissue composite preservation solution, the composition of the auxiliary preservation solution is:

The composition of the auxiliary preservation solution is:

Histidine 1-100 mmol/L

Tryptophan 1-3 mmol/L

Glutamine 1-3 mmol/L

Glutathione 4-6 mmol/L

Adenosine triphosphate 1-10 mmol/L

Deferoxamine 5-50 umol/L

Insulin 30-50 IU/L

Hydrocortisone 40-60 mg/L.

The hair follicle tissue composite preservation solution described in the above technical solution, wherein: the composition of the solute in the basic preservation solution is:

Sodium chloride 20 mmol/L

Potassium chloride 15 mmol/L

Magnesium chloride hexahydrate 5 mmol/L

Lacturonic acid 50 mmol/L

Trehalose 13 mmol/L

Mannitol 30 mmol/L

Potassium dihydrogen phosphate 28 mmol/L.

The hair follicle tissue composite preservation solution described in the above technical solution, wherein: the auxiliary preservation solution is composed of:

Histidine 40 mmol/L

Tryptophan 3 mmol/L

Glutamine 1 mmol/L

Glutathione 6 mmol/L

Adenosine triphosphate 6 mmol/L

Deferoxamine 28 umol/L

Insulin 35 IU/L

Hydrocortisone 45 mg/L.

The hair follicle tissue composite preservation solution described in the above technical scheme, wherein: the osmotic pressure of the hair follicle tissue composite preservation solution is 310mmol/L ± 10mmol/L.

The hair follicle tissue composite preservation solution described in the above technical solution, wherein: the auxiliary preservation solution component is selected from histidine, tryptophan, glutamine, glutathione, adenosine triphosphate, deferoxamine, insulin and hydrocortisone one or more of.

The hair follicle tissue composite preservation solution described in the above technical solution, wherein: the auxiliary preservation solution component is selected from histidine, tryptophan, glutamine, glutathione, adenosine triphosphate, deferoxamine, insulin and hydrocortisone one or more of.

The preparation method of the hair follicle tissue composite preservation solution described in the above technical scheme, wherein, the method comprises the following steps:

Step 1: Take sodium chloride, potassium chloride, magnesium chloride hexahydrate, lacturonic acid, trehalose, mannitol and potassium dihydrogen phosphate as solutes, and add the above solutes into sterile medical It is configured into a solution with a total volume of 500mL in water;

Step 2: Add the components of the auxiliary preservation solution to the solution prepared in Step 1, and mix them evenly; add medical sterile water to make a solution with a total volume of 1000 mL, and adjust the pH to 7.4 with sodium hydroxide/hydrochloric acid to obtain composite preservation wherein the content of auxiliary preservation solution components is calculated by the volume of hair follicle tissue composite preservation solution 1000mL;

Step 3: filter and sterilize the composite preservation solution obtained in step 2 with a bacterial filter to obtain a hair follicle tissue composite preservation solution.

A kind of preservation method that maintains hair follicle in vitro activity, is characterized in that, described preservation method comprises the following steps:

(1) Preservation of hair follicles in vitro: take the composite preservation solution of hair follicle tissue, pre-cool at 4°C; take the unit hair follicles obtained by hair follicle extraction, rinse with Ringer's solution to remove blood clots and impurities; put the rinsed unit hair follicles in the cold It is fully immersed in the composite preservation solution of hair follicle tissue, and the preservation temperature should be maintained at 4 °C;

(2) Hair follicle transplantation in vivo: remove the hair follicle tissue composite preservation solution, rinse the unit hair follicle with Ringer's solution, and then transplant into the body after removing the residual preservation solution;

The hair follicle tissue composite preservation solution is the hair follicle tissue composite preservation solution described in the above technical solution.

The present invention has the following beneficial effects:

1. All the components of the present invention are commercial reagents that can be used in clinical practice, and there are no risky suspicious components. The preserved hair follicles can be directly used for clinical transplantation treatment after washing with simple saline, which is safe and effective, and has a good clinical application prospect.

2. The components of the present invention do not contain colloids with high viscosity, and all components except mannitol and lacturonic acid are natural physiological substrates, which provide a good cell environment for the preservation of hair follicles in vitro. Its osmotic pressure component is close to the level of intracellular fluid, which can effectively prevent cell edema and dehydration without affecting the nervous system. Low concentration of sodium and potassium ions can effectively avoid intracellular ion confusion. Does not contain calcium ions, can avoid intracellular calcium overload, reduce low temperature-induced changes in mitochondrial permeability and the resulting apoptosis. The contained phosphate buffer pair can effectively prevent the metabolic acidosis of cells caused by ischemia and hypoxia. Tryptophan is an effective cell membrane stabilizer, mannitol is an osmotic diuretic, and lacturonic acid is an impermeable substance, both of which can effectively inhibit cell edema under cryopreservation. Trehalose preferentially combines with water molecules in the solution, reducing the content of free water in the solution, lowering the freezing point, and effectively reducing the formation of ice crystals in a low-temperature storage environment. As antioxidants, histidine, glutamine and glutathione can effectively prevent the accumulation of reactive oxygen species in cells and cause damage to cells. Adenosine triphosphate is an energy substrate, and a large amount of energy can be released when hydrolyzed. Insulin acts as a potent regulator of metabolic function, promoting cellular utilization of glucose and amino acids. Hydrocortisone promotes the growth of epithelial type cells and is effective in maintaining hair follicle growth. Insulin and hydrocortisone are essential nutrients for hair follicle culture in vitro. Adding the preservation solution can effectively improve the hypoxia state and increase the activity of cells in the hair follicle. Deferoxamine is an iron complexing agent, which can reduce the damage of intracellular chelatable iron ions to the cytoplasmic membrane and reduce the level of lipid peroxidation in the plasma membrane.

Description of drawings:

1. Figure 1 is a comparison diagram of the results of live and dead staining of unit hair follicle cells and the results of dead cell counts after the hair follicles of the unit hair follicles are stored in different preservation solutions of Comparative Examples 1-4 and Example 1 of the present invention, respectively.

2. Fig. 2 is a comparison chart of the proliferation/apoptosis staining results and counting results of unit hair follicle cells after storage of unit hair follicles with different preservation solutions of Examples 1-3 of the present invention respectively.

3. Fig. 3 is the experimental result of the growth of hair follicle organ cultured hair follicles after the unit hair follicles are stored with different preservation solutions of Comparative Example 1-2 and Example 1-3 of the present invention respectively.

4. Figure 4 shows the detection results of the microscopic and ultrastructural levels of the hair follicle tissue after the unit hair follicles were stored with different preservation solutions in Comparative Example 1 and Example 1 of the present invention.

Detailed ways:

In order to facilitate understanding of the present invention, the present invention will be described more fully below, and preferred embodiments of the present invention will be given. However, the present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that a thorough and complete understanding of the present disclosure is provided.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terms used herein in the description of the present invention are for the purpose of describing specific embodiments only, and are not intended to limit the present invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Embodiment 1: a kind of composite preservation solution of hair follicle tissue and the preservation method of maintaining the activity of isolated hair follicles:

1. A hair follicle tissue composite preservation solution of the present embodiment is composed of a basic preservation solution and an auxiliary preservation solution, wherein the basic preservation solution is composed of a solute, a solvent and a pH value regulator;

The pH adjuster is sodium hydroxide/hydrochloric acid, which is used to adjust the pH of the hair follicle tissue composite preservation solution to 7.4;

The solvent is medical sterile water; a part of the solvent is used to dissolve the solute to form a solution, and the volume of the solution is 1/2 of the volume of the composite preservation solution of the hair follicle tissue; the other part of the solvent is used to add the auxiliary preservation solution to the volume of the solution to the volume of hair follicle tissue composite preservation solution;

The preparation process of hair follicle tissue composite preservation solution is as follows:

Step 1: take sodium chloride, potassium chloride, magnesium chloride hexahydrate, lacturonic acid, trehalose, mannitol and potassium dihydrogen phosphate as solutes, add the above solutes into the solvent medical sterile water to prepare a solution with a total volume of 500mL, The content of each component in the solute is calculated as the volume of 1000 mL of hair follicle tissue composite preservation solution:

Sodium chloride 20 mmol/L

Potassium chloride 15 mmol/L

Magnesium chloride hexahydrate 5 mmol/L

Lacturonic acid 50 mmol/L

Trehalose 13 mmol/L

Mannitol 30 mmol/L

Potassium dihydrogen phosphate 28 mmol/L;

Step 2: Add the auxiliary preservation solution components histidine, tryptophan, glutamine, glutathione, adenosine triphosphate, deferoxamine, insulin and hydrocortisone to the solution prepared in step 1, and mix well ; Add solvent medical sterile water to make a solution with a total volume of 1000mL, and sodium hydroxide/hydrochloric acid is adjusted to pH 7.4 to obtain a composite preservation solution; wherein the content of each component of the auxiliary preservation solution is a solution with a volume of 1000mL of the hair follicle tissue composite preservation solution The volume is calculated as:

Histidine 40 mmol/L

Tryptophan 3 mmol/L

Glutamine 1 mmol/L

Glutathione 6 mmol/L

Adenosine triphosphate 6 mmol/L

Deferoxamine 28 umol/L

Insulin 35 IU/L

Hydrocortisone 45 mg/L;

Step 3: filter and sterilize the composite preservation solution obtained in step 2 with a bacterial filter to obtain a hair follicle tissue composite preservation solution.

2. The steps of the preservation method for maintaining the activity of isolated hair follicles are as follows:

(1) Preservation of hair follicles in vitro: take the composite preservation solution of hair follicle tissue, pre-cool at 4°C; take the unit hair follicles obtained by hair follicle extraction, rinse with Ringer's solution to remove blood clots and impurities; put the rinsed unit hair follicles in the cold It is fully immersed in the composite preservation solution of hair follicle tissue, and the preservation temperature should be maintained at 4 °C;

(2) Transplantation of hair follicles in vivo: remove the composite preservation solution of hair follicle tissue, rinse unit hair follicles with Ringer's solution, and then transplant into the body after removing the residual preservation solution.

Embodiment 2: a kind of composite preservation solution of hair follicle tissue and the preservation method of maintaining the activity of isolated hair follicles:

The content of each component of the solute in the present embodiment is:

Sodium chloride 5 mmol/L

Potassium chloride 10 mmol/L

Magnesium chloride hexahydrate 1 mmol/L

Lacturonic acid 25 mmol/L

Trehalose 5 mmol/L

Mannitol 1 mmol/L

Potassium dihydrogen phosphate 5 mmol/L

The content of each component in the auxiliary preservation solution in the present embodiment is:

Histidine 1 mmol/L

Tryptophan 1 mmol/L

Glutamine 1 mmol/L

Glutathione 1 mmol/L

Adenosine triphosphate 1 mmol/L

Deferoxamine 5 umol/L

Insulin 3 IU/L

Hydrocortisone 4 mg/L

The preparation method of the hair follicle tissue composite preservation solution and the preservation method for maintaining the activity of the isolated hair follicles in this embodiment are the same as those in Embodiment 1, and the difference is only in the content of the components, which will not be repeated in detail.

Embodiment 3: a kind of hair follicle tissue composite preservation solution and the preservation method that maintains the activity of isolated hair follicles:

The content of each component of the solute in this example is:

Sodium chloride 30 mmol/L

Potassium chloride 20 mmol/L

Magnesium chloride hexahydrate 10 mmol/L

Lacturonic acid 75 mmol/L

Trehalose 20 mmol/L

Mannitol 50 mmol/L

Potassium dihydrogen phosphate 50 mmol/L

The content of each component in the auxiliary preservation solution in the present embodiment is:

Histidine 100 mmol/L

Tryptophan 20 mmol/L

Glutamine 20 mmol/L

Glutathione 50 mmol/L

Adenosine triphosphate 50 mmol/L

Deferoxamine 50 umol/L

Insulin 50 IU/L

Hydrocortisone 60 mg/L

The preparation method of the hair follicle tissue composite preservation solution and the preservation method for maintaining the activity of the isolated hair follicles in this embodiment are the same as those in Embodiment 1, and the difference is only in the content of the components, which will not be repeated in detail.

Comparative Example 1:

The conventional preservative solution configuration on the market is Ringer's solution.

Comparative Example 2:

This comparative example is the same as the above-mentioned Example 1, except that it does not add auxiliary preservation solution components (ie, histidine, tryptophan, glutamine, glutathione, adenosine triphosphate, desferrioxamine, insulin and hydrocortisone). pine) hair follicle tissue based preservation solution.

Comparative Example 3:

This comparative example is the same as the above-mentioned Example 1, except that it is a composite hair follicle tissue preservation solution that only adds part of the amino acid nutritional components of the auxiliary preservation solution (ie, histidine, tryptophan, glutamine, and glutathione).

Comparative Example 4:

This comparative example is the same as the above-mentioned Example 1, except that it only adds part of the amino acid nutritional components of the auxiliary preservation solution (ie histidine, tryptophan, glutamine and glutathione) and the supplementary components of cellular energy metabolism (adenosine triphosphate, insulin and hydrocortisone) compound hair follicle tissue preservation solution.

result:

1. Use Comparative Examples 1-4 and Example 1 to preserve unit hair follicles for 6 hours, and then perform live and dead cell staining and dead cell counting of unit hair follicles.

Live and dead cell staining: Take A bottle of ethidium dimer-1 (excitation wavelength of 545nm, red fluorescent dead cells can be seen under a fluorescence microscope) and B bottle of calcein-AM (excitation wavelength of 490 ± 10nm, Viable cells with green fluorescence can be seen under a fluorescence microscope), and mix the two thoroughly. Take the unit hair follicles to be treated and add an appropriate amount of DMEM/F12 medium for immersion, then add an equal amount of living and dead staining mixed reagent, mix well, and keep at room temperature for 20 minutes in the dark. After rinsing with PBS, the hair follicles were taken and observed under an inverted fluorescence microscope, and photographed and recorded.

The results are shown in FIG. 1 , the number of apoptotic cells in Example 1 is the lowest among the control examples 1-4, and the protection effect for unit hair follicles is the best. Compared with Comparative Example 1, the apoptosis of hair follicle cells after resuscitation is reduced in Comparative Example 2, indicating that the basic preservation solution of the present invention has a better protective effect on hair follicle cells, which is better than the Ringer's solution commonly used in clinical practice. . Compared with Comparative Example 2, the apoptosis of hair follicle cells after resuscitation was further reduced, indicating that the amino acid nutritional components (histidine, tryptophan, glutamine and glutathione) of the auxiliary preservation solution according to the present invention. ) can effectively improve the anti-apoptotic ability and avoid cell damage caused by reactive oxygen species. Compared with Comparative Example 3, the apoptosis of hair follicle cells after resuscitation was further reduced, indicating that the supplementary components of energy metabolism in the auxiliary preservation solution (adenosine triphosphate, deferoxamine, insulin and hydrocortisone) can provide nutrients on the basis of nutrition. Synergistically improve the anti-apoptotic ability of hair follicle cells. Compared with Comparative Example 4, the apoptosis of hair follicle cells after resuscitation was significantly reduced in Example 1, indicating that the complete auxiliary preservation solution components are histidine, tryptophan, glutamine, glutathione, adenosine triphosphate, deferoxamine , insulin and hydrocortisone can synergistically improve the anti-apoptotic ability of hair follicle cells, and exert the greatest protective effect of the preservation solution on isolated hair follicle cells.

2. Use Examples 1-3 to store the hair follicles for 6 hours, and detect the proliferation (Ki67)/apoptosis (TUNEL) staining results and counting results of the hair follicle cells.

Ki67 staining: Frozen sections of hair follicle tissue to be detected were fixed with acetone at -20°C for 10 min, washed twice with PBS, and blocked with 10% serum homologous to the secondary antibody for 20 min. Ki67 primary antibody (rabbit anti-human) was added dropwise to the slices, overnight at 4°C; secondary antibody (goat anti-rabbit, FITC-labeled) was added dropwise, 37°C, 45min, washed with PBS for 3 times, DAB color development. The number of Ki67-positive cells in the section was observed under an upright fluorescence microscope, and photographed and recorded.

TUNEL staining: Frozen sections of hair follicle tissue to be tested were fixed with acetone at -20°C for 10min, rinsed twice with PBS, dripped with lProteinaseK working solution on each sample, and reacted at 37°C for 15-20min; dropwise added the prepared DNase I reaction solution , treated at 37°C for 30min; add 50µl Streptavidin-TRITC labeling solution dropwise, put it in a wet box, and react at 37°C in the dark for 30min; The number of TUNEL-positive cells in the section was observed under an upright fluorescence microscope, and photographed and recorded.

The results are shown in Figure 2. It was shown by cell proliferation/apoptosis staining that Example 1 significantly reduced the apoptotic rate of hair follicle cells (TUNEL+) and increased its proliferative activity (Ki67+), reflecting Example 1 and Example 1. Compared with 1-2, it can better maintain the activity of isolated hair follicles.

3. Use Comparative Examples 1-2 and Examples 1-3 to preserve unit hair follicles for 6 hours, and then conduct in vitro hair follicle organ culture experiments.

Hair follicle organ culture: After rinsing the treated hair follicles with PBS, they were placed in a 24-well plate separately, and Williams'E medium was added, 500ul per well, overnight at 37°C. On the second day, the hair follicles with poor morphology or in the degenerative phase were removed, and the culture medium was replaced every 3 days. The shape of hair follicles, the length of hair shaft growth and the photographing records were observed under a stereoscopic microscope.

The results are shown in FIG. 3 , Example 1 significantly prolonged the hair shaft growth length, and the growth phase ratio was increased. This shows from the organ level that Example 1 can better maintain the activity of the isolated hair follicles.

4. The unit hair follicles were preserved for 6 hours using Comparative Example 1 and Example 1, respectively, and the hair follicles were detected at the histological level.

HE staining: The hair follicle tissue to be detected was fixed with 4% paraformaldehyde and routinely sectioned. The dried tissue pieces were sequentially treated with xylene I-xylene II-xylene III (10 min each)-100% ethanol-95% ethanol-80% ethanol-50% ethanol-deionized water (5 min each). Eosin was added dropwise to the tissue pieces to observe the coloration of the tissue, followed by rinsing with running water. Hematoxylin was added dropwise to the tissue pieces, and the staining of the tissue was observed, followed by rinsing with running water. Then observe under the upright microscope, take pictures and record.

Transmission electron microscope detection: The hair follicle tissue to be detected was fixed in 2.5% glutaraldehyde and post-fixed in 1% osmium tetroxide. After dehydration with gradient concentrations of acetone, tissue sections were performed. The tissue pieces were dried with a critical desiccator, and conductively treated by sputtering in a vacuum. It was then placed in a scanning electron microscope for observation, and photographed for recording.

The results are shown in Figure 4. Compared with Comparative Example 1, Example 1 reduced the intracellular and interstitial edema and mitochondrial swelling of hair follicles in vitro by H&E staining and transmission electron microscopy, and was beneficial to maintain the dermal sheath and collagen. The normal structure of fibers. (ORS: outer root sheath tissue, IRS: inner root sheath tissue)

The above are only preferred embodiments of the present invention, and do not limit the present invention in any form or substance. Those skilled in the art, without departing from the scope of the technical solution of the present invention, can use the above The technical content disclosed, and the equivalent changes made with some changes, modifications and evolutions are equivalent embodiments of the present invention; at the same time, any equivalent changes made to the above embodiments according to the essential technology of the present invention , modification and evolution, all still belong to the scope of the technical solution of the present invention.

Claims (6)

1. a hair follicle tissue composite preservation solution, is characterized in that: the hair follicle tissue composite preservation solution is made up of a basic preservation solution and an auxiliary preservation solution, and wherein the basic preservation solution is made up of a solute, a solvent and a pH value regulator; The pH adjuster is sodium hydroxide/hydrochloric acid, which is used to adjust the pH of the hair follicle tissue composite preservation solution to 7.4; The solvent is medical sterile water; a part of the solvent is used to dissolve the solute to form a solution, and the volume of the solution is 1/2 of the volume of the composite preservation solution of the hair follicle tissue; the other part of the solvent is used to add the auxiliary preservation solution to the volume of the solution to the volume of hair follicle tissue composite preservation solution; In terms of the volume of hair follicle tissue composite preservation solution, the composition of the solute is: Sodium chloride 5-30 mmol/L Potassium chloride 10-20 mmol/L Magnesium chloride hexahydrate 1-10 mmol/L Lacturonic acid 25-75 mmol/L Trehalose 5-20 mmol/L Mannitol 10-50 mmol/L Potassium dihydrogen phosphate 5-50 mmol/L In terms of the volume of hair follicle tissue composite preservation solution, the composition of the auxiliary preservation solution is: The composition of the auxiliary preservation solution is: Histidine 1-100 mmol/L Tryptophan 1-3 mmol/L Glutamine 1-3 mmol/L Glutathione 4-6 mmol/L Adenosine triphosphate 1-10 mmol/L Deferoxamine 5-50 umol/L Insulin 30-50 IU/L Hydrocortisone 40-60 mg/L. 2. hair follicle tissue composite preservation solution according to claim 1, is characterized in that: the composition of solute in described basic preservation solution is: Sodium chloride 20 mmol/L Potassium chloride 15 mmol/L Magnesium chloride hexahydrate 5 mmol/L Lacturonic acid 50 mmol/L Trehalose 13 mmol/L Mannitol 30 mmol/L Potassium dihydrogen phosphate 28 mmol/L. 3. hair follicle tissue composite preservation solution according to claim 1, is characterized in that: the composition of described auxiliary preservation solution is: Histidine 40 mmol/L Tryptophan 3 mmol/L Glutamine 1 mmol/L Glutathione 6 mmol/L Adenosine triphosphate 6 mmol/L Deferoxamine 28 umol/L Insulin 35 IU/L Hydrocortisone 45 mg/L. 4. the hair follicle tissue composite preservation solution according to any one of claims 1-3, is characterized in that: the osmotic pressure of the hair follicle tissue composite preservation solution is 310mmol/L ± 10mmol/L. 5. the preparation method of the hair follicle tissue composite preservation solution described in any one of claim 1-4, wherein, described method comprises the steps: Step 1: Take sodium chloride, potassium chloride, magnesium chloride hexahydrate, lacturonic acid, trehalose, mannitol and potassium dihydrogen phosphate as solutes, and add the above solutes into sterile medical It is configured into a solution with a total volume of 500mL in water; Step 2: Add the components of the auxiliary preservation solution to the solution prepared in Step 1, and mix them evenly; add medical sterile water to make a solution with a total volume of 1000 mL, and adjust the pH to 7.4 with sodium hydroxide/hydrochloric acid to obtain composite preservation wherein the content of auxiliary preservation solution components is calculated by the volume of hair follicle tissue composite preservation solution 1000mL; Step 3: filter and sterilize the composite preservation solution obtained in step 2 with a bacterial filter to obtain a hair follicle tissue composite preservation solution. 6. a preservation method that maintains hair follicle in vitro activity, is characterized in that, described preservation method comprises the steps: (1) Preservation of hair follicles in vitro: take the composite preservation solution of hair follicle tissue, pre-cool at 4°C; take the unit hair follicles obtained by hair follicle extraction, rinse with Ringer's solution to remove blood clots and impurities; put the rinsed unit hair follicles in the cold It is fully immersed in the composite preservation solution of hair follicle tissue, and the preservation temperature should be maintained at 4 °C; (2) Hair follicle transplantation in vivo: remove the hair follicle tissue composite preservation solution, rinse the unit hair follicle with Ringer's solution, and then transplant into the body after removing the residual preservation solution; The hair follicle tissue composite preservation solution is the hair follicle tissue composite preservation solution of any one of claims 1-4.

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