CN111937862B - Testis tissue cryopreservation method based on single seminiferous tubule perfusion - Google Patents
Testis tissue cryopreservation method based on single seminiferous tubule perfusion Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
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- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention relates to a testis tissue cryopreservation method based on single seminiferous tubule perfusion, which comprises the following steps: firstly, preparing testis tissues into a single seminiferous tubule, then placing the single seminiferous tubule in a cryopreservation protective agent 1 for soaking for 10min, then using a cryopreservation protective agent 2 to soak and perfuse the seminiferous tubule, realizing vitrification cooling and simultaneously reducing the osmotic damage of the protective agent to the maximum extent, then quickly transferring the perfused seminiferous tubule to a Cryo-pick single sperm cryopreservation sheet, directly soaking the seminiferous tubule in liquid nitrogen for quick cooling after being sleeved in the Cryo-tube, and quickly placing the seminiferous tubule in a resuscitation solution for soaking after cooling. Compared with the traditional testis cell suspension cryopreservation method, the method has the advantages that: (1) the complete structure of the seminiferous tubule is reserved; (2) effectively reducing the oxidative stress level of testicular cells; (3) the death of a large number of spermatogonial cells and supporting cells after cryopreservation is effectively reduced; (4) the recovery rate and activity of sperms in the seminiferous tubules are high.
Description
Technical Field
The invention relates to the technical field of pill tissue cryopreservation, in particular to a testis tissue cryopreservation method based on single seminiferous tubule perfusion, namely, a small amount of testis tissue is subjected to vitrification cryopreservation after a cryopreservation liquid is perfused into the seminiferous tubule.
Background
Currently, fertility of reproductive age population shows an overall decline trend in the global scope, WHO human reproduction special planning administration reports that the infertility rate of global population is at least 15%, the number of pairs of global infertility couples is about 6000-. According to the investigation report of the sterile status of Chinese infertility published by the population association, the rate of the sterile of China is as high as 15-20%, and the number of pairs of the sterile couples is about 2000 ten thousand. The etiology is about 60% of the disease originated from female factors, while in the male aspect, the total number of sperms is reduced by more than half in 50 years (the density of the sperms is reduced to be more than or equal to 6000 ten thousand/ml, and is reduced to be more than or equal to 1500 ten thousand/ml), and moreover, the problem of the reduction of the quality of the sperms of the human is also worried, and the incidence rate of azoospermia is about 1%.
Most azoospermia belongs to non-obstructive azoospermia caused by the damage of spermatic function of testis, at present, the treatment means for azoospermia mainly comprises endocrine treatment, operation treatment and the like, wherein the endocrine treatment can lead about 10 percent of azoospermia patients to spontaneously discharge sperms, and the sperms can be directly stored by intracytoplasmic sperm microinjection technology (ICSI) or by the rare sperm freezing technology. However, most patients fail to spontaneously discharge sperm by endocrine therapy, and for such patients, microscopic testicular spermiation surgery is the last treatment option. Some patients with incomplete loss of spermatogenic function have seminiferous tubules remaining in the testicular tissue with intact spermatogenesis, which are much coarser and denser than the spermatitless seminiferous tubules under the field of surgical microscopy, called "foci of spermatogenesis", usually only in a very small area of the testicle. After the spermatogenic foci are taken out in operation, spermatogenic cells in the spermatogenic tubules are released by mechanical stripping and shearing, and the spermatogenic cells are purified by methods such as gradient density centrifugation and the like and then subjected to ICSI or cryopreservation.
The above surgical approaches do not address the underlying cause of patient dysspermia. Currently, the in vitro culture expansion and induced differentiation of spermatogonial stem cells into functional spermatozoa is the most promising therapeutic approach for reestablishing fertility in patients with NOA. In vivo spermatogenesis depends on a complex spermatogenic microenvironment, including a seminiferous tubule structure formed by various somatic cells and a regulation network formed by various signal analysis. At present, because the in vivo spermatogenesis microenvironment is not fully understood, the efficiency of an in vitro culture system is still low, and the in vitro culture system is not enough for clinical treatment. Meanwhile, as the 'spermatogenic focus' is not renewable, the spermatogenic disorder of the NOA patient generally progresses progressively, so that a set of freezing method for preserving a very small amount of testicular seminiferous tubule tissues obtained by an operation needs to be established.
At present, the cryopreservation method of male testicular tissues/cells mainly comprises testicular cell suspension cryopreservation and slow cooling cryopreservation of testicular tissues. In the former, testis tissues are separated into cell suspension by mechanical cutting and enzyme digestion, so that the space structure of spermatogenesis is destroyed, and the influence on in vitro culture is certainly generated. The latter is mainly used for freezing and storing non-rare testis tissues at present, and large sharp ice crystals are easily generated in the freezing and storing process, and the seminiferous tubule structure is also damaged. The recently developed tissue and organ vitrification freezing technology can reduce the damage of ice crystal, and high concentration of cryoprotectant and fast temperature lowering rate are required to reach glass state in the temperature lowering process. The seminiferous tubules have a relatively closed lumen, and it is difficult to achieve the desired concentration of fluid in the lumen by direct immersion of cryoprotectants, and in addition, extended cryoprotectant loading increases osmotic damage of the cryoprotectant to testicular tissue/cells. Based on these problems, we developed a cryopreservation method for single seminiferous tubule (rare testicular tissue) by first perfusing cryoprotectants into the seminiferous tubule and then performing vitrification cooling, which is used for preserving fertility of related male patients, and no relevant report is found at present about the related content of the invention.
Disclosure of Invention
The invention aims to provide a cryopreservation method based on vitrification cooling after perfusion of single seminiferous tubule cryopreservation liquid, which is used for preserving complete spermatogenesis environment and preserving fertility of related male patients.
In order to achieve the purpose, the invention adopts the technical scheme that:
firstly, the invention provides a testis tissue cryopreservation method based on single seminiferous tubule perfusion, which comprises the following steps:
(1) preparing testis tissue into single seminiferous tubule;
soaking the single seminal tubule in a cryopreservation protective agent 1 for 10min, wherein the cryopreservation protective agent 1 consists of the following components in concentration: 10% DMSO, 10% FBS and 0.1M sucrose;
then placing the single seminiferous tubule into a cryopreservation protection 2 to be soaked for 1min, and simultaneously pouring the cryopreservation protection 2 into the single seminiferous tubule, wherein the cryopreservation protection agent 2 consists of the following components in concentration: 20% DMSO, 20% FBS and 0.5M sucrose;
rapidly transferring the perfused and soaked single seminiferous tubule to a Cryo-piece single sperm cryopreservation sheet, directly immersing the Cryo-piece single sperm cryopreservation sheet into liquid nitrogen for cooling, and sequentially and respectively immersing the single seminiferous tubule in a resuscitation solution 1 for 1min and in a resuscitation solution 2 for 3min after cooling, wherein the resuscitation solution 1 consists of the following components in concentration: 10% FBS and 1M sucrose, and the resuscitation solution 2 is composed of the following components in concentration: 10% FBS, 0.5M sucrose and DF 12;
and finally transferring to a conventional culture condition.
Preferably, the step (1) comprises the following steps: the testis tissue is removed from the stroma and is stuck and spread into a single seminiferous tubule by using a microscopic instrument, and then the single seminiferous tubule is cut into pieces with the length less than or equal to 1 cm.
Preferably, in the step (3), the cryopreservation protection 2 is poured into the single seminiferous tubule at the speed of 1ul/s, and the injection amount is 1-2 ul/cm.
Preferably, the resuscitation fluid 1 and resuscitation fluid 2 in step (4) are pre-treated to 37 ℃ before use.
Preferably, the conventional culture conditions in step (5) include: DF12 medium with 10% FBS.
Preferably, the source of testicular tissue is a patient who has not had a complete loss of spermatogenic function and has a seminiferous tubule remaining in which spermatogenesis can occur intact.
Secondly, the invention also provides a testis tissue cryopreservation reagent combination, which comprises the following components:
(1) cryopreservation protective agent 1: consists of 10% DMSO, 10% FBS and 0.1M sucrose; and
(2) freezing and storing protective agent 2: consisting of 20% DMSO, 20% FBS and 0.5M sucrose; and
(3) 1, resuscitation solution: consists of 10% FBS and 1M sucrose; and
(4) and (3) resuscitation solution 2: consists of 10% FBS, 0.5M sucrose and DF 12.
Furthermore, the invention also provides application of the reagent in preparation of a testis tissue cryopreservation kit.
Furthermore, the invention also provides a kit for cryopreservation of testicular tissue, the kit comprises the reagent combination and an instruction for using the kit, and the instruction for using the kit comprises the method.
Preferably, the testicular tissue source is a patient who has not had a complete loss of spermatogenic function and has a seminiferous tubule remaining in which spermatogenesis can occur intact.
The invention has the advantages that:
1. compared with the traditional testis cell suspension frozen storage, the invention reserves the complete structure of the seminiferous tubule for possible seminiferous cell culture in the future.
2. Compared with the traditional slow cooling and freezing storage of the testicular tissue mass, the invention disperses the seminiferous tubules into single pieces, can obviously reduce the formation of large-volume sharp ice crystals in testicular tissue gaps, and can better avoid the integrity of the seminiferous tubules from being damaged by the ice crystals (the result can be shown in figure 3).
3. According to the invention, the cryoprotectant is filled in the single nerve tubule, so that the testis tissue is prevented from contacting hypertonic solution for a long time before being cooled, the osmotic injury and the chemical toxicity injury of the cryoprotectant are reduced, and the oxidative stress level of testis cells is effectively reduced (the result can be shown in figure 4).
4. The invention avoids the formation of ice crystals in cells by rapid cooling vitrification cryopreservation, and can effectively reduce the death of a large number of spermatogonial stem cells and supporting cells after cryopreservation (the result can be shown in figure 5).
5. The recovery rate and activity of the sperms in the seminiferous tubules cryopreserved by the method of the invention are not obviously different from the straw vitrification freezing method which is clinically and conventionally used at present (the result can be seen in figure 6).
In conclusion, the method preferably selects various steps, the type and concentration of the cryopreservation protective agent, the optimal cryopreservation protection loading time, the optimal concentration of the resuscitation solution and the method, can better preserve the seminiferous tubule structure and maintain important somatic cells, spermatogonial stem cells and mature sperms of spermatogenesis, provides a new treatment scheme for patients without complete loss of spermatogenesis function, and can achieve the purpose of preserving the fertility of related male patients.
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FIG. 1 is a main flow diagram of the process of the present invention.
FIG. 2 is a comparison of the freezing preservation effect of different formulations of freezing preservation protective agent in the method of the invention.
FIG. 3 is a comparison of ice crystal formation and seminiferous tubules in the method of the present invention compared to conventional methods of cryopreservation of testicular tissue.
FIG. 4 is a comparison of oxidative stress levels at different concentrations and soaking times.
FIG. 5 shows the comparison of apoptosis in the method of the present invention and the conventional method of cryopreservation of testicular tissue.
FIG. 6 is a comparison of sperm cell parameters for the present invention method and current methods of sperm cryopreservation.
Detailed Description
The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the disclosure of the present invention, and equivalents fall within the scope of the appended claims.
Example 1 testis tissue cryopreservation reagent combination
The method comprises the following steps: cryopreservation protective agent 1: consists of 10% DMSO, 10% FBS and 0.1M sucrose; and cryopreservation protectant 2: consisting of 20% DMSO, 20% FBS and 0.5M sucrose; and a resuscitation fluid 1: consists of 10% FBS and 1M sucrose; and a resuscitation fluid 2: consists of 10% FBS, 0.5M sucrose and DF 12.
EXAMPLE 2 cryopreservation protectant infusion of Single seminiferous tubules
Isolated testis tissues obtained in a microscopic semen collection operation of a non-obstructive azoospermia patient who is seen in a first people hospital in Shanghai city are immediately put into HTF liquid with the volume at least 20 times that of the testis tissues and transported to a laboratory. During the period, 10ul of the cryopreservation protective agent 2 is sucked into an ICSI glass fine needle and connected with a microfluidic pump. And (3) when the sample is transported to a laboratory, putting the sample into a small dish filled with HTF liquid, and slightly separating the direct interstitium of the seminiferous tubules under a stereoscope by using a pair of microscissors and a pair of microscopical forceps to enable the seminiferous tubules to become single seminiferous tubules. Seminiferous tubules over 1cm in length were cut to 1cm using microshearing. Transferring the seminiferous tubules into a small dish filled with the frozen protective agent 1, and soaking for 10min on a shaking table. And then transferring the seminiferous tubules into the cryopreservation protective agent 2, immediately injecting the cryopreservation protective agent 2 in the glass needle into the seminiferous tubules at the speed of 1ul/s under the visual field of 15 times of magnification of a stereoscopic mirror, wherein the total injection amount is 1-2 ul of cryopreservation liquid per 1cm of the seminiferous tubules, and the loading time of the seminiferous tubules in the cryopreservation protective agent 2 is 1 min.
Example 3 cryopreservation and Resuscitation of Single seminiferous tubule
And taking the single seminiferous tubule out of the cryopreservation protective agent 2, slightly sucking the cryopreservation protective agent on the surface by using absorbent paper, then placing the cryoprotectant on a Cryo-piece single sperm cryopreservation sheet, directly immersing the Cryo-piece single sperm cryopreservation sheet into liquid nitrogen for rapid cooling after sleeving the Cryo-piece single sperm cryopreservation sheet, and moving the cryopreservation sample to a corresponding storage position in the liquid nitrogen after the temperature is stabilized. Before resuscitation, the resuscitation fluid is heated to 37 ℃, the seminiferous tubules are taken out of liquid nitrogen, then are quickly placed in the resuscitation fluid 1 (10% FBS +1M sucrose) for soaking for 1min, then are placed in the resuscitation fluid 2 (10% FBS +0.5M sucrose + DF123min) for soaking for 3min, and finally are transferred to appropriate culture conditions (for example, 10% FBS DF12 culture medium).
Example 4 control experiment
1 method of experiment
Dividing the in vitro testis tissue in the operation into a plurality of groups, wherein each group is frozen and preserved according to different methods respectively, and comprises a normal group, a low-concentration non-perfusion group, a low-concentration perfusion group, a high-concentration non-perfusion group, a high-concentration perfusion group, a single-root perfusion method freezing and preserving group, a traditional slow-rate freezing and preserving group, a low-concentration slow-rate group, a high-concentration slow-rate group, a low-concentration single-root group, a high-concentration single-root group, a low-concentration soaking 5min group, a low-concentration soaking 15min group, a high-concentration soaking 5min group, a high-concentration soaking 15min group, a low-concentration vitrification perfusion group, a high-concentration vitrification perfusion group, a fumigating quick freezing group and a vitrification perfusion group.
Cryopreservation protectant 1 used in the experiment: the low concentration means that: 10% DMSO + 10% FBS +0.1M sucrose; the high concentration means: 20% DMSO + 10% FBS +0.1M sucrose. Cryopreservation protectant used in the experiment 2: the low concentration means that: 20% DMSO + 20% FBS +0.5M sucrose; the high concentration means: 40% DMSO + 20% FBS +0.5M sucrose.
2 results of the experiment
2.1 comparison of the cryopreservation effects of cryoprotectants of different formulations in the method
As shown in FIG. 2, the region of the tissue in which ice crystals are formed during cooling is more refractive and appears as a dark region. The low concentration perfusion group retains the complete structure of the seminiferous tubules compared with the traditional slow method cryopreservation group.
2.2 this method compares ice crystal formation and seminiferous tubules with the traditional methods for cryopreservation of testis tissue
FIG. 3, wherein A, B shows interstitial ice crystal formation during cryopreservation by various methods; c-graph represents lumen integrity based on H & E staining statistics; d-graph shows lumen integrity based on SMA protein immunofluorescence staining statistics. Compared with the traditional slow cooling and freezing storage of the testicular tissue mass, the method provided by the invention disperses the seminiferous tubules into single pieces, can obviously reduce the formation of large-volume sharp ice crystals in testicular tissue gaps, and can better avoid the integrity of the seminiferous tubules from being damaged by the ice crystals.
2.3 comparison of oxidative stress levels at different concentrations and soaking times
See FIG. 4, fluorescence intensity for use
The cell oxidative stress detection kit detects an average fluorescence signal in a standard area of a single seminiferous tubule, and the fluorescence intensity is in direct proportion to the cell oxidative stress level. The result shows that the cryoprotectant is filled in the single nerve tubule of the low-concentration group of 5min, so that the testis tissue is prevented from contacting the hypertonic solution for a long time before being cooled, the osmotic damage and the chemical toxicity damage of the cryoprotectant are reduced, and the oxidative stress level of the testis cells is effectively reduced.
2.4 comparison of apoptosis between different concentrations (vitrification infusion group) and the conventional testis tissue cryopreservation method
As shown in FIG. 5, apoptosis was labeled with c-PARP antibody and spermatogenic cells were labeled with DDX4 antibody. The results show that the low-concentration vitrified perfusion group has the largest number of cells, the largest number of non-apoptotic cells and the smallest apoptosis ratio except the normal group. The reason is attributed to the fact that the rapid cooling vitrification cryopreservation of the low-concentration vitrification perfusion group avoids the formation of ice crystals in cells, and can effectively reduce the death of a large number of spermatogonial stem cells and supporting cells after cryopreservation.
2.5 comparison of sperm cell parameters of the present invention with sperm cell cryopreservation methods (fumigation rapid freezing set)
Referring to fig. 6, the recovery rate and activity of the sperm in the seminiferous tubules frozen by the method of the present invention are not significantly different from those of the straw vitrification freezing method which is currently and clinically used.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and additions can be made without departing from the principle of the present invention, and these should also be considered as the protection scope of the present invention.
Claims (10)
1. A testis tissue cryopreservation method based on single seminiferous tubule perfusion is characterized by comprising the following steps:
(1) preparing testis tissue into single seminiferous tubule;
(2) soaking the single seminal tubule in a cryopreservation protective agent 1 for 10min, wherein the cryopreservation protective agent 1 consists of the following components in concentration: 10% DMSO, 10% FBS and 0.1M sucrose;
(3) then placing the single seminiferous tubule into a cryopreservation protection 2 to be soaked for 1min, and simultaneously pouring the cryopreservation protection 2 into the single seminiferous tubule, wherein the cryopreservation protection agent 2 consists of the following components in concentration: 20% DMSO, 20% FBS and 0.5M sucrose;
(4) rapidly transferring the perfused and soaked single seminiferous tubule to a single sperm cryopreservation sheet, directly immersing the sheet into Cryo-tubule, then cooling the sheet, and sequentially and respectively immersing the single seminiferous tubule in a resuscitation solution 1 for 1min and a resuscitation solution 2 for 3min after cooling, wherein the resuscitation solution 1 comprises the following components in concentration: 10% FBS and 1M sucrose, and the resuscitation solution 2 is composed of the following components in concentration: 10% FBS, 0.5M sucrose and DF 12;
(5) and finally transferring to a conventional culture condition.
2. The method according to claim 1, wherein the step (1) is as follows: the testis tissue is removed from the stroma and is stuck and spread into a single seminiferous tubule by using a microscopic instrument, and then the single seminiferous tubule is cut into pieces with the length less than or equal to 1 cm.
3. The method according to claim 1, wherein the cryopreservation protection 2 is injected into the single seminiferous tubule at a rate of 1ul/s in the step (3), and the injection amount is 1-2 ul/cm.
4. The method of claim 1, wherein the resuscitation fluid 1 and resuscitation fluid 2 in step (4) are pre-treated to 37 ℃ prior to use.
5. The method according to claim 1, wherein the conventional culture conditions in step (5) comprise: DF12 medium with 10% FBS.
6. The method of claim 1 wherein the source of testicular tissue is a patient who has not had a complete loss of spermatogenic function and has residual seminiferous tubules that allow sperm to develop intact.
7. A testis tissue cryopreservation reagent combination, comprising:
(1) cryopreservation protective agent 1: consists of 10% DMSO, 10% FBS and 0.1M sucrose; and
(2) freezing and storing protective agent 2: consisting of 20% DMSO, 20% FBS and 0.5M sucrose; and
(3) 1, resuscitation solution: consists of 10% FBS and 1M sucrose; and
(4) and (3) resuscitation solution 2: consists of 10% FBS, 0.5M sucrose and DF 12.
8. Use of a combination of reagents according to claim 7 for the preparation of a kit for cryopreservation of testicular tissue.
9. A kit for cryopreservation of testicular tissue comprising the combination of reagents of claim 7 and instructions for use of the kit comprising the method of claim 1.
10. The kit of claim 9 wherein the testicular tissue source is a patient who has not had a complete loss of spermatogenic function and has residual seminiferous tubules that enable sperm production to occur intact.
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| Title |
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| Cryopreservation of Rat Seminiferous Tubules Using Biopolymers and Slow Non-Controlled Rate Cooling;N.O. Volkova 等;《Probl Cryobiol Cryomed》;20181231;第28卷(第4期);278-292 * |
| Exposure of Seminiferous Tubules of Immature Rats to Cryoprotective Media of Various Compositions;N.O. Volkova 等;《Probl Cryobiol Cryomed》;20171231;第27卷(第3期);203-218 * |
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