Vitrified cryopreservation and resuscitation method of hair follicles

Abstract

The invention belongs to the technical field of tissue cryopreservation, and discloses a hair follicle cryopreservation and resuscitation method which comprises a cryopreservation step and a resuscitation step, and the cryopreservation step comprises the steps of soaking hair follicles in equilibrium liquid, taking out the hair follicles, soaking the hair follicles in a cryopreservation tube containing cryopreservation liquid and cryopreserving the hair follicles; the resuscitation step comprises the steps of taking out the frozen hair follicles from the freezing tube, and soaking the frozen hair follicles into resuscitation liquid. The vitrification cryopreservation method provided by the invention is simple in procedure, short in time consumption and low in cost, avoids ice crystal growth processes such as crystallization, anti-vitrification and ice crystal regrowth in a slow-freezing cryopreservation process, can reduce cryopreservation damage of the hair follicles, and improves the survival rate of the resuscitated hair follicles.

Description

Technical Field

[0001] The invention belongs to the technical field of tissue cryopreservation, and in particular relates to a vitrification cryopreservation and revival method of hair follicles. Background Art

[0002] Androgenic alopecia (AGA) is the most common hair loss disease in the world, with more than 200 million patients in my country. It is mainly manifested by the progressive miniaturization of hair follicles in the forehead and top of the head until they fall out, eventually leading to baldness. Due to the side effects, insufficient efficacy and lifelong medication of drug treatments (finasteride and minoxidil), autologous hair transplantation has become the most effective treatment to solve the appearance of baldness caused by AGA and rebuild the frontal hairline.

[0003] However, the hair follicles in the non-transplanted areas of the patient will still fall off over time, and will need to be transplanted again after 10 to 20 years to improve the appearance. When hair is transplanted again, on the one hand, large-scale hair loss caused by disease progression will lead to insufficient number of hair follicles in the donor area. On the other hand, aging is accompanied by follicle aging, resulting in a low survival rate of transplanted hair follicles. In this regard, some scholars have proposed a concept: extracting and vitrifying the frontal hair follicles that have not yet been miniaturized when people at high risk of hair loss are young can increase the number of hair follicle donors; in addition, transplanting high-quality hair follicles stored in their youth for older patients can improve the survival rate of transplanted hair follicles. Therefore, providing a cryopreservation solution suitable for hair follicles can solve the problems of low transplant survival rate of aged hair follicles and insufficient number of hair follicles in the donor area faced by autologous hair transplantation.

[0004] However, hair follicles are micro-organs composed of multiple germ layers and multiple types of cells. Their structural complexity will limit the penetration of cryoprotectants (CPA), and different cells have different sensitivities to CPA. Therefore, the freezing of hair follicles is much more difficult than that of single cells. According to public domestic and foreign data, there is currently only one patent involving the field of hair follicle cryopreservation, which provides a hair follicle vitrification cryopreservation solution (invention patent CN114946829A), but it has the following defects: 1) It can only maintain the cell activity of hair follicles during the cryopreservation process. The revived hair follicles can be used to extract hair papilla cells, dermal sheath cells and outer root sheath cells, but the tissue activity and structural functional integrity of the hair follicles are destroyed. The revived hair follicles cannot grow normally and be used for hair transplantation. 2) The patent only discloses the composition of the cryopreservation solution, and does not mention the freezing and resuscitation methods of hair follicles. 3) Its composition contains 10% fetal bovine serum, which not only has a heterologous immune source of unknown composition, but also has potential safety risks during clinical application. And studies have shown that culture medium containing serum will inhibit the activity of hair follicles in vitro culture, which may be because the serum contains transforming growth factor and molecules with certain specific effects.

[0005] In summary, there is currently no suitable method for freezing and resuscitation of hair follicles, and the existing hair follicle freezing solution cannot maintain the complete tissue structure and function of hair follicles. Therefore, it is urgent to establish a freezing and resuscitation method that can maintain the integrity of the hair follicle tissue structure and function, so that the hair follicles can not only be used for cell extraction after long-term storage, but also grow normally to meet the needs of hair transplantation surgery. Summary of the invention

[0006] In order to overcome the defects of the prior art, the present invention provides a hair follicle vitrification and thawing method. Compared with slow freezing, the vitrification method used in the present invention has a simple procedure, short time consumption, low cost, avoids the ice crystal growth process including crystallization, devitrification and ice crystal regrowth in the slow freezing process, can reduce the freezing damage of hair follicles, and improve the survival rate of hair follicles after thawing.

[0007] The first aspect of the present invention aims to provide a method for freezing and reviving hair follicles.

[0008] The purpose of the second aspect of the present invention is to provide application of the method of the first aspect of the present invention in cryopreservation and resuscitation of tissues.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is:

[0010] The first aspect of the present invention provides a method for freezing and reviving hair follicles, comprising a freezing step and a reviving step, wherein:

[0011] The freezing step includes: soaking the hair follicles in a balancing solution, taking out the hair follicles, soaking them in a freezing tube containing a freezing solution, and freezing them;

[0012] The resuscitation steps include: taking out the frozen hair follicles from the cryopreservation tube and immersing them in a resuscitation solution.

[0013] In some embodiments of the present invention, the balancing solution includes balancing solution A, balancing solution B and balancing solution C, and the balancing solution A, balancing solution B or balancing solution C is obtained by mixing a base solution and a freezing solution in different volume ratios.

[0014] In some embodiments of the present invention, the balancing solution A, balancing solution B or balancing solution C is obtained by mixing the base solution and the freezing solution in a volume ratio of 2 to 3:1, 1:1, or 1:2 to 3.

[0015] In some preferred embodiments of the present invention, the balancing solution A, balancing solution B or balancing solution C is obtained by mixing the base solution and the freezing solution in a volume ratio of 3:1, 1:1, or 1:3.

[0016] In some embodiments of the present invention, the freezing solution comprises dimethyl sulfoxide, ethylene glycol, human serum albumin, sucrose, hydrocortisone, insulin, glutamine, adenosine triphosphate, melatonin, epigallocatechin gallate, resveratrol and culture medium.

[0017] In some embodiments of the present invention, the serum-free vitrification solution includes 15-25 v / v% dimethyl sulfoxide, 15-25 v / v% ethylene glycol, 2-3 mmol / L human serum albumin, 0.5-2.5 mol / L sucrose, 0.01-1 mmol / L hydrocortisone, 0.2-20 mol / L insulin, 1-4 mmol / L glutamine, 0.1-20 mmol / L adenosine triphosphate, 2-8 mmol / L melatonin, 0.4-10 mmol / L epigallocatechin gallate, 0.4-10 mmol / L resveratrol and culture medium.

[0018] In some preferred embodiments of the present invention, the serum-free vitrification solution includes 18-20 v / v% dimethyl sulfoxide, 18-20 v / v% ethylene glycol, 2.1-2.7 mmol / L human serum albumin, 0.5-2 mol / L sucrose, 0.05-0.1 mmol / L hydrocortisone, 1-2 mol / L insulin, 1-3 mmol / L glutamine, 1-5 mmol / L adenosine triphosphate, 5-8 mmol / L melatonin, 1-5 mmol / L epigallocatechin gallate, 1-5 mmol / L resveratrol and culture medium.

[0019] In some embodiments of the present invention, the base solution includes hydrocortisone, insulin, glutamine, adenosine triphosphate, melatonin, epigallocatechin gallate and resveratrol.

[0020] In some embodiments of the present invention, the basic solution includes 0.01-1 mmol / L of hydrocortisone, 0.2-20 mol / L of insulin, 1-4 mmol / L of glutamine, 0.1-20 mmol / L of adenosine triphosphate, 2-8 mmol / L of melatonin, 0.4-10 mmol / L of epigallocatechin gallate, 0.4-10 mmol / L of resveratrol and culture medium.

[0021] In some preferred embodiments of the present invention, the basic solution includes 0.05-0.1 mmol / L of hydrocortisone, 1-2 mol / L of insulin, 1-3 mmol / L of glutamine, 1-5 mmol / L of adenosine triphosphate, 5-8 mmol / L of melatonin, 1-5 mmol / L of epigallocatechin gallate, 1-5 mmol / L of resveratrol and culture medium.

[0022] In some embodiments of the present invention, the hair follicles are soaked in balancing solution A, balancing solution B and balancing solution C in sequence, and the soaking time is 10 to 30 min, 10 to 30 min and 5 to 25 min, respectively.

[0023] In some preferred embodiments of the present invention, the hair follicles are soaked in balancing solution A, balancing solution B and balancing solution C in sequence, and the soaking time is 10-15 min, 10-20 min and 5-15 min in sequence.

[0024] In some more preferred embodiments of the present invention, the hair follicles are soaked in balancing solution A, balancing solution B and balancing solution C in sequence, and the soaking time is 10-15 min, 10-15 min and 5-10 min in sequence.

[0025] In some optimal embodiments of the present invention, the hair follicles are soaked in balancing solution A, balancing solution B and balancing solution C in sequence, and the soaking time is 15 min, 10 min and 5 min in sequence.

[0026] In some embodiments of the present invention, the hair follicles are immersed in the cryopreservation tube containing the cryopreservation solution for 5 to 20 minutes.

[0027] In some preferred embodiments of the present invention, the hair follicles are immersed in the cryopreservation tube containing the cryopreservation solution for 5 to 10 minutes.

[0028] In some more preferred embodiments of the present invention, the hair follicles are immersed in the cryopreservation tube containing the cryopreservation solution for 5 minutes.

[0029] In some embodiments of the present invention, the resuscitation fluid includes resuscitation fluid A, resuscitation fluid B and resuscitation fluid C, and the resuscitation fluid A and resuscitation fluid B include human serum albumin, sucrose, hydrocortisone, insulin, glutamine, adenosine triphosphate and culture medium; the resuscitation fluid C includes human serum albumin, hydrocortisone, insulin, glutamine, adenosine triphosphate and culture medium.

[0030] In some embodiments of the present invention, the resuscitation fluid A includes 0.9-1.5 mmol / L human serum albumin, 0.25-1 mol / L sucrose, 0.01-1 mmol / L hydrocortisone, 0.2-20 mol / L insulin, 1-3 mmol / L glutamine, 0.17-17 mmol / L adenosine triphosphate and culture medium.

[0031] In some embodiments of the present invention, the resuscitation fluid B includes 0.9-1.5 mmol / L human serum albumin, 0.125-0.5 mol / L sucrose, 0.01-1 mmol / L hydrocortisone, 0.2-20 mol / L insulin, 1-3 mmol / L glutamine, 0.17-17 mmol / L adenosine triphosphate and the remainder of culture medium.

[0032] In some embodiments of the present invention, the resuscitation fluid C includes 0.9-1.5 mmol / L human serum albumin, 0.01-1 mmol / L hydrocortisone, 0.2-20 mol / L insulin, 1-3 mmol / L glutamine, 0.17-17 mmol / L adenosine triphosphate and the remainder of culture medium.

[0033] In some embodiments of the present invention, the culture medium is Walliam's E medium. Walliam's E medium contains a variety of inorganic salts, amino acids and vitamins. Compared with other culture media such as DMEM, its ingredients are most suitable for the growth needs of hair follicles and are suitable for in vitro culture of hair follicles.

[0034] In some embodiments of the present invention, the frozen hair follicles are immersed in resuscitation solution A, resuscitation solution B and resuscitation solution C in sequence; the immersion time is 5 to 20 min, 5 to 20 min and 5 to 20 min in sequence.

[0035] In some preferred embodiments of the present invention, the soaking time is 5 to 15 min, 5 to 15 min and 5 to 15 min, respectively.

[0036] In some more preferred embodiments of the present invention, the soaking time is 5 to 10 min, 5 to 10 min and 10 to 15 min, respectively.

[0037] In some optimal embodiments of the present invention, the soaking time is 5 minutes, 10 minutes and 15 minutes respectively.

[0038] In some embodiments of the present invention, in the resuscitation step, the cryopreservation tube containing the hair follicles is placed in a 37-40° C. water bath for rewarming before being immersed in the resuscitation solution.

[0039] In some embodiments of the present invention, in the resuscitation step, the hair follicles are taken out after being soaked in the resuscitation solution, eluted with an elution solution, and then rinsed with physiological saline or compound sodium chloride injection.

[0040] In some embodiments of the present invention, the eluent is a culture medium containing human serum albumin.

[0041] In some embodiments of the present invention, the eluent is Walliam's E medium containing 1-2 mmol / L human serum albumin.

[0042] In some embodiments of the present invention, the elution time of the eluent is 4 to 8 minutes.

[0043] The second aspect of the present invention provides application of the method of the first aspect of the present invention in cryopreservation and resuscitation of tissues.

[0044] In some embodiments of the present invention, the tissues include but are not limited to liver, kidney, skin, tumor, hair follicle and other tissues.

[0045] The beneficial effects of the present invention are:

[0046] The method provided by the present invention fills the gaps in hair follicle cryopreservation and resuscitation methods, serum-free hair follicle cryopreservation fluid, and hair follicle resuscitation fluid. Compared with slow cryopreservation, the vitrification cryopreservation procedure adopted by the present invention is simple, time-saving, and low-cost. More importantly, the slow cryopreservation process will include ice crystal growth processes such as crystallization, devitrification, and ice crystal regrowth. The growth of ice crystals will cause fatal mechanical damage to tissues and organs. The essence of vitrification cryopreservation is that the liquid forms a highly viscous state during the freezing and solidification process, and the movement of molecules is highly constrained. There are no crystals or only a small amount of crystals inside. It is considered to be the only promising technology for long-term preservation of large tissues and organs. The survival rate of hair follicle tissue after resuscitation is over 94%, and in vitro culture shows that its activity is no different from that of fresh hair follicles. It can be used for clinical transplantation treatment or hair follicle mesenchymal stem cell extraction. It is safe and effective and has good clinical application prospects.

[0047] The method provided by the present invention uses functional balancing solution, freezing solution, resuscitation solution, etc. containing energy metabolism regulators (hydrocortisone, insulin, glutamine, ATP) and antioxidants (melatonin, epigallocatechin gallate, resveratrol), which can effectively reduce the cell damage caused by metabolism and redox imbalance caused by insufficient synthesis of adenosine triphosphate (ATP) and the massive production of reactive oxygen species (ROS) in the freezing and thawing process of hair follicle tissue, and provide certain nutrients, thereby maintaining the cryopreservation viability of hair follicles. Hydrocortisone, insulin and ATP can protect the dermal components of hair follicles (hair follicle mesenchymal stem cells); melatonin can protect the epidermal components of hair follicles (hair follicle stem cells and hair matrix cells). There is no need to add unsafe ingredients such as animal-derived serum, which reduces the possibility of animal pathogen contamination; the combined use of non-permeable and permeable cryoprotectants can inhibit ice crystal formation and reduce the cytotoxic effect of permeable cryoprotectants. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1It is the time used in the hair follicle cryopreservation and thawing method of Examples 1 to 9.

[0049] Figure 2 The following is a comparison chart of the survival rate results of the hair follicles after cryopreservation and resuscitation treatment in Example 1 and Examples 8-9 and fresh hair follicles (i.e., Control Example 1) in in vitro culture. In the figure, ns represents P>0.05, ** represents P<0.01, *** represents P<0.005, and **** represents P<0.001.

[0050] Figure 3 The hair follicles are elongated after the cryopreservation and resuscitation treatments of Example 1 and Comparative Examples 6 to 8, and fresh hair follicles (ie, Control Example 1) were cultured in vitro for 5 days.

[0051] Figure 4 The figures are comparison diagrams of the morphological results of the hair follicles after cryopreservation and resuscitation treatments of Example 1 and Example 2 and fresh hair follicles (ie, Control Example 1) at the 0th, 1st, 3rd and 5th in vitro cultures. DETAILED DESCRIPTION

[0052] The present invention is further described in detail below through specific examples.

[0053] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0054] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0055] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0056] All embodiments and comparative examples used the following freezing solution, balancing solution, resuscitation solution and elution solution:

[0057] Basic solution: hydrocortisone 0.1mmol / L, insulin 2mol / L, glutamine 2mmol / L, adenosine triphosphate 1.7mmol / L, melatonin 5mmol / L, catechin gallate 2mmol / L, resveratrol 2mmol / L and the balance of Walliam's E medium.

[0058] Cryopreservation solution: dimethyl sulfoxide 20v / v%, ethylene glycol 20v / v%, human serum albumin 2.4mmol / L, sucrose 1mol / L, hydrocortisone 0.1mmol / L, insulin 2mol / L, glutamine 2mmol / L, adenosine triphosphate 1.7mmol / L, melatonin 5mmol / L, catechin gallate 2mmol / L, resveratrol 2mmol / L and the balance of Walliam's E medium.

[0059] Balanced solution A, balanced solution B and balanced solution C: the basal solution and the freezing solution were mixed in volume ratios of 3:1, 1:1 and 1:3, respectively.

[0060] Resuscitation fluid A: human serum albumin 1mmol / L, sucrose 1mol / L, hydrocortisone 0.1mmol / L, insulin 2mol / L, glutamine 2mmol / L, adenosine triphosphate 1.7mmol / L and the balance of Walliam's E medium.

[0061] Resuscitation solution B: human serum albumin 1mmol / L, sucrose 0.5mol / L, hydrocortisone 0.1mmol / L, insulin 2mol / L, glutamine 2mmol / L, adenosine triphosphate 1.7mmol / L and the balance of Walliam's E medium.

[0062] Resuscitation fluid C: human serum albumin 1mmol / L, hydrocortisone 0.1mmol / L, insulin 2mol / L, glutamine 2mmol / L, adenosine triphosphate 1.7mmol / L and the balance of Walliam's E medium.

[0063] Eluent: 1 mmol / L human serum albumin and the remainder of Walliam's E medium.

[0064] Example 1

[0065] A method for freezing and resuscitating hair follicles comprises the following steps:

[0066] (1) Hair follicle cryopreservation: The hair follicles were obtained and rinsed with normal saline or compound sodium chloride injection, and then immersed in balancing solution A, balancing solution B, and balancing solution C in turn for 15 min, 10 min, and 5 min, respectively; the hair follicles were taken out, immersed in a cryopreservation tube containing cryopreservation solution for 5 min, and then directly placed in liquid nitrogen for long-term storage.

[0067] (2) Hair follicle resuscitation: The frozen hair follicles are removed from the cryopreservation tube, placed in a 37-40°C water bath to rewarm, and then immersed in resuscitation solutions A, B, and C in sequence for 5 min, 10 min, and 15 min, respectively; the hair follicles are placed in the elution solution for 5 min, and then rinsed with normal saline or compound sodium chloride injection to complete the resuscitation.

[0068] Example 2

[0069] A method for freezing and resuscitating hair follicles comprises the following steps:

[0070] (1) Hair follicle cryopreservation: The hair follicles were obtained and rinsed with normal saline or compound sodium chloride injection, and then immersed in balancing solution A, balancing solution B, and balancing solution C in turn for 20 min, 15 min, and 10 min, respectively; the hair follicles were removed and immersed in a cryopreservation tube containing cryopreservation solution for 10 min, and then directly placed in liquid nitrogen for long-term storage.

[0071] (2) Hair follicle resuscitation: The frozen hair follicles are removed from the cryopreservation tube, placed in a 37-40°C water bath to rewarm, and then immersed in resuscitation solutions A, B, and C in sequence for 5 min, 10 min, and 15 min, respectively; the hair follicles are placed in the elution solution for 5 min, and then rinsed with normal saline or compound sodium chloride injection to complete the resuscitation.

[0072] Example 3

[0073] A method for freezing and resuscitating hair follicles comprises the following steps:

[0074] (1) Hair follicle cryopreservation: The hair follicles were obtained and rinsed with normal saline or compound sodium chloride injection, and then immersed in balancing solution A, balancing solution B, and balancing solution C in turn for 25 min, 20 min, and 15 min, respectively; the hair follicles were removed and immersed in a cryopreservation tube containing cryopreservation solution for 15 min, and then directly placed in liquid nitrogen for long-term storage.

[0075] (2) Hair follicle resuscitation: The frozen hair follicles are removed from the cryopreservation tube, placed in a 37-40°C water bath to rewarm, and then immersed in resuscitation solutions A, B, and C in sequence for 5 min, 10 min, and 15 min, respectively; the hair follicles are placed in the elution solution for 5 min, and then rinsed with normal saline or compound sodium chloride injection to complete the resuscitation.

[0076] Example 4

[0077] A method for freezing and resuscitating hair follicles comprises the following steps:

[0078] (1) Hair follicle cryopreservation: The hair follicles were obtained and rinsed with normal saline or compound sodium chloride injection, and then immersed in balancing solution A, balancing solution B, and balancing solution C in turn for 25 min, 25 min, and 25 min, respectively; the hair follicles were removed and immersed in a cryopreservation tube containing cryopreservation solution for 15 min, and then directly placed in liquid nitrogen for long-term storage.

[0079] (2) Hair follicle resuscitation: The frozen hair follicles are removed from the cryopreservation tube, placed in a 37-40°C water bath to rewarm, and then immersed in resuscitation solutions A, B, and C in sequence for 5 min, 10 min, and 15 min, respectively; the hair follicles are placed in the elution solution for 5 min, and then rinsed with normal saline or compound sodium chloride injection to complete the resuscitation.

[0080] Example 5

[0081] A method for freezing and resuscitating hair follicles comprises the following steps:

[0082] (1) Hair follicle cryopreservation: The hair follicles were obtained and rinsed with normal saline or compound sodium chloride injection. The hair follicles were then immersed in a cryopreservation tube containing cryopreservation solution for 5 minutes and then directly placed in liquid nitrogen for long-term storage.

[0083] (2) Hair follicle resuscitation: The frozen hair follicles are removed from the cryopreservation tube, placed in a 37-40°C water bath to rewarm, and then immersed in resuscitation solutions A, B, and C in sequence for 5 min, 10 min, and 15 min, respectively; the hair follicles are placed in the elution solution for 5 min, and then rinsed with normal saline or compound sodium chloride injection to complete the resuscitation.

[0084] Example 6

[0085] A method for freezing and resuscitating hair follicles comprises the following steps:

[0086] (1) Hair follicle cryopreservation: The hair follicles were obtained and rinsed with normal saline or compound sodium chloride injection, and then immersed in balancing solution A, balancing solution B, and balancing solution C in turn for 25 min, 20 min, and 15 min, respectively; the hair follicles were removed and immersed in a cryopreservation tube containing cryopreservation solution for 15 min, and then directly placed in liquid nitrogen for long-term storage.

[0087] (2) Hair follicle resuscitation: The frozen hair follicles are removed from the cryopreservation tube and placed in a 37-40°C water bath to rewarm. The hair follicles are then eluted directly with elution solution for 5 minutes without being immersed in the resuscitation solution. They are then rinsed with normal saline or compound sodium chloride injection to complete the resuscitation.

[0088] Example 7

[0089] A method for freezing and resuscitating hair follicles comprises the following steps:

[0090] (1) Hair follicle cryopreservation: The hair follicles were obtained and rinsed with normal saline or compound sodium chloride injection, and then immersed in balancing solution A, balancing solution B, and balancing solution C in turn for 25 min, 25 min, and 25 min, respectively; the hair follicles were removed and immersed in a cryopreservation tube containing cryopreservation solution for 15 min, and then directly placed in liquid nitrogen for long-term storage.

[0091] (2) Hair follicle resuscitation: The frozen hair follicles are removed from the cryopreservation tubes, placed in a 37-40°C water bath for rewarming, and then immersed in resuscitation solutions A, B, and C in sequence for 20 min, 20 min, and 20 min, respectively; the hair follicles are placed in the elution solution for 5 min, and then rinsed with normal saline or compound sodium chloride injection to complete the resuscitation.

[0092] Example 8

[0093] A method for freezing and resuscitating hair follicles comprises the following steps:

[0094] (1) Hair follicle cryopreservation: The hair follicles were obtained and rinsed with normal saline or compound sodium chloride injection, and then immersed in balancing solution A, balancing solution B, and balancing solution C in turn for 25 min, 25 min, and 25 min, respectively; the hair follicles were removed and immersed in a cryopreservation tube containing cryopreservation solution for 15 min, and then directly placed in liquid nitrogen for long-term storage.

[0095] (2) Hair follicle resuscitation: The frozen hair follicles are removed from the cryopreservation tube, placed in a 37-40°C water bath to rewarm, and then immersed in resuscitation solutions A, B, and C in sequence for 10 min, 15 min, and 20 min, respectively; the hair follicles are placed in the elution solution for 5 min, and then rinsed with normal saline or compound sodium chloride injection to complete the resuscitation.

[0096] Example 9

[0097] A method for freezing and resuscitating hair follicles comprises the following steps:

[0098] (1) Hair follicle cryopreservation: The hair follicles were obtained and rinsed with normal saline or compound sodium chloride injection, and then immersed in balancing solution A, balancing solution B, and balancing solution C in turn for 25 min, 25 min, and 25 min, respectively; the hair follicles were removed and immersed in a cryopreservation tube containing cryopreservation solution for 15 min, and then directly placed in liquid nitrogen for long-term storage.

[0099] (2) Hair follicle resuscitation: The frozen hair follicles are removed from the cryopreservation tube, placed in a 37-40°C water bath to rewarm, and then immersed in resuscitation solutions A, B, and C in sequence for 5 min, 5 min, and 5 min, respectively; the hair follicles are placed in the elution solution for 5 min, and then rinsed with normal saline or compound sodium chloride injection to complete the resuscitation.

[0100] The time used in the hair follicle cryopreservation and thawing methods of Examples 1 to 9 Figure 1 shown.

[0101] Effect Example

[0102] Fresh hair follicles were cryopreserved for 3 months using the hair follicle cryopreservation and thawing methods of Examples 1 to 9, and then thawed using the corresponding hair follicle thawing methods, followed by hair follicle organ culture. After 5 days, the survival rate and hair follicle elongation were calculated. Fresh hair follicles were used as a control example, wherein the hair follicle organ culture:

[0103] (1) Twelve hair follicles were selected from each group and gently transferred into a disposable 24-well plate in a clean bench, with one hair follicle per well. 0.5 mL of Williams E medium was added for suspension culture. The cells were cultured in an incubator at 37°C and 5% CO2 for 5 days.

[0104] (2) Take photos under a stereoscope on days 0, 1, 3, and 5, count the length of the hair follicles, and calculate the total elongation over 5 days;

[0105] (3) On the 5th day, the survival rate of hair follicles was counted.

[0106] The results of hair follicle survival rate and hair follicle elongation are as follows Figure 2 to Figure 4 shown.

[0107] The survival rate and elongation of Example 4 and Example 5 were both 0, indicating that in this cryopreservation scheme, directly immersing in liquid nitrogen after adding the cryopreservation solution cannot be used for the cryopreservation of hair follicle tissue. In addition, the equilibrium time during the cryopreservation process is too long, and the toxicity of the cryoprotectant will cause the hair follicles to be inactivated.

[0108] Compared with Examples 2 to 3, the hair follicle activity and hair follicle growth ability of Example 1 are significantly increased, indicating that the equilibrium time is suitable for the cryopreservation and recovery ability of hair follicle tissue, and the effect is related to the length of the equilibrium time. The cryopreservation effect is best at the equilibrium time of Example 1.

[0109] The survival rate and elongation of Example 6 were both 0, indicating that the traditional resuscitation scheme of taking out the cryotube from liquid nitrogen and eluting directly after rewarming in a water bath cannot be used for the resuscitation of frozen hair follicle tissue.

[0110] Compared with Examples 7 to 9, the survival rate and 5-day elongation after resuscitation in Example 1 were both improved, indicating that the resuscitation effect of hair follicle tissue is related to the resuscitation time, and the resuscitation time in Example 1 is most suitable for the resuscitation of frozen hair follicle tissue.

[0111] The survival rate and 5-day elongation of Example 1 were the highest among the above examples, and the protective effect on hair follicles was the best. The survival rate of hair follicles after resuscitation was as high as 94%, which was not significantly different from the control group.

[0112] The results are as follows Figure 4 As shown, the morphological changes of hair follicles in the in vitro culture process between Example 1 and Example 4 also show that Example 1 has high protection for hair follicles, can maintain cell activity and resist apoptosis, and protect the integrity of hair follicle tissue, further confirming the rationality and effectiveness of the hair follicle freezing solution of the present invention.

[0113] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for freezing and reviving hair follicles, comprising a freezing step and a reviving step, wherein: The freezing step includes: soaking the hair follicles in a balancing solution, taking out the hair follicles, soaking them in a cryopreservation tube containing the freezing solution, and freezing them; the resuscitation step includes: taking out the frozen hair follicles from the cryopreservation tube, and soaking them in the resuscitation solution.

2. The method according to claim 1, characterized in that The balancing solution includes balancing solution A, balancing solution B and balancing solution C, wherein the balancing solution A, balancing solution B or balancing solution C is obtained by mixing a base solution and a freezing solution in different volume ratios; preferably, the freezing solution includes dimethyl sulfoxide, ethylene glycol, human serum albumin, sucrose, hydrocortisone, insulin, glutamine, adenosine triphosphate, melatonin, epigallocatechin gallate, resveratrol and culture medium; Preferably, the base solution comprises hydrocortisone, insulin, glutamine, adenosine triphosphate, melatonin, epigallocatechin gallate and resveratrol.

3. The method according to claim 2, characterized in that The hair follicles are soaked in balancing solution A, balancing solution B and balancing solution C in sequence, with the soaking time being 10 to 30 minutes, 10 to 30 minutes and 5 to 25 minutes in sequence.

4. The method according to claim 1, characterized in that The hair follicles are immersed in the cryopreservation tube containing the cryopreservation solution for 5 to 20 minutes.

5. The method according to any one of claims 1 to 4, characterized in that: The resuscitation fluid includes resuscitation fluid A, resuscitation fluid B and resuscitation fluid C. The resuscitation fluid A and resuscitation fluid B include human serum albumin, sucrose, hydrocortisone, insulin, glutamine, adenosine triphosphate and culture medium; the resuscitation fluid C includes human serum albumin, hydrocortisone, insulin, glutamine, adenosine triphosphate and culture medium.

6. The method according to claim 5, characterized in that The frozen hair follicles are sequentially immersed in resuscitation solution A, resuscitation solution B and resuscitation solution C; the immersion time is 5 to 20 minutes, 5 to 20 minutes and 5 to 20 minutes respectively.

7. The method according to claim 2, characterized in that: The freezing solution comprises 15-25 v / v% dimethyl sulfoxide, 15-25 v / v% ethylene glycol, 2-3 mmol / L human serum albumin, 0.5-2.5 mol / L sucrose, 0.01-1 mmol / L hydrocortisone, 0.2-20 mol / L insulin, 1-4 mmol / L glutamine, 0.1-20 mmol / L adenosine triphosphate, 2-8 mmol / L melatonin, 0.4-10 mmol / L epigallocatechin gallate, 0.4-10 mmol / L resveratrol and culture medium.

8. The method according to claim 2, characterized in that: The basic liquid comprises 0.01-1 mmol / L of hydrocortisone, 0.2-20 mol / L of insulin, 1-4 mmol / L of glutamine, 0.1-20 mmol / L of adenosine triphosphate, 2-8 mmol / L of melatonin, 0.4-10 mmol / L of epigallocatechin gallate, 0.4-10 mmol / L of resveratrol and culture medium.

9. The method according to claim 7 or 8, characterized in that: The balancing solution A, balancing solution B or balancing solution C is obtained by mixing the base solution and the freezing solution in a volume ratio of 2 to 3:1, 1:1, or 1:2 to 3.

10. Use of the method according to any one of claims 1 to 9 in cryopreservation and resuscitation of tissues.

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