CN111345284A - Human testicular tissue suspension cryoprotectant and preparation method thereof - Google Patents
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Abstract
The invention discloses a human testicular tissue suspension cryoprotectant and a preparation method thereof, wherein the human testicular tissue suspension cryoprotectant comprises the following raw materials: sodium chloride, potassium chloride, calcium chloride, magnesium chloride, potassium dihydrogen phosphate, sodium bicarbonate, HEPES, caffeine, sodium pyruvate, pentoxifylline, glycine, glucose, and sucrose; the human testicular tissue suspension cryoprotectant further comprises sodium lactate and glycerol. The invention also comprises a preparation method of the human testicular tissue suspension cryoprotectant and a freezing method adopting the human testicular tissue suspension cryoprotectant. The invention specially designs the cryoprotectant aiming at the testicular tissue suspension, can improve the testicular sperm freezing recovery effect, effectively solves the problems of potential safety hazard, poor freezing recovery effect, difficulty in finding movable testicular sperm and the like, and has very important clinical application value.
Description
Technical Field
The invention relates to the technical field of cryoprotectants and preparation thereof, in particular to a human testicular tissue suspension cryoprotectant and a preparation method thereof.
Background
With the development of social industrialization, environmental pollution is more serious, the infertility proportion is more and more, the incidence rate of couples in the childbearing age is up to 9.3 percent, the male factors account for about 30 to 50 percent, and the azoospermia patients account for 10 to 20 percent of the male factors. In 1992, the advent of intracytoplasmic sperm injection (ICSI) technology realized the desire of male oligozoospermia patients to produce offspring, and in 1993, Schoysman applied ICSI to testicular sperm lines and succeeded, bringing good news to the clinical application of subsequent testicular sperm. Clinically, a patient with azoospermia generally needs to perform testicular diagnostic biopsy on the basis of eliminating genetic diseases, the result can judge whether sperms exist in the testicles, and when the testicular sperms exist in the diagnostic biopsy, the testicular sperms can be selected to be frozen for later use in an auxiliary reproductive cycle.
Two major technical factors of sperm freezing are a freezing method and a cryoprotectant, and the selection of the cryoprotectant is carried out according to the characteristics of frozen sperm. The motility of human sperm is obtained after the sperm enters into epididymis and undergoes a series of morphological structure, biochemical metabolism and physiological function changes, and the sperm in testis is basically mature from the morphological and structural aspects but has no motility. Therefore, compared with the conventional semen freezing, the testicular sperm freezing has the characteristics of small quantity, weak activity, immobility or weak vibrating sperm.
At present, no cryoprotectant specially aiming at testicular tissues exists in the market, most common cryoprotectants are used for freezing, the cryoprotectant mainly has the function of reducing the formation of ice crystals in the freezing process so that sperms smoothly pass through the critical temperature, and the cryoprotectant is mainly divided into two categories of permeable cryoprotectants and non-permeable cryoprotectants. The existing cryoprotectant in the market mainly contains biogenic substances such as yolk, albumin and the like, and has certain hidden danger in use safety. The common cryoprotectant is used for freezing, and due to the special use of non-testicular sperms and the characteristics of testicular sperms, the common cryoprotectant has the defects of poor freezing effect and incapability of finding movable testicular sperms for ICSI after recovery.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a human testicular tissue suspension cryoprotectant and a preparation method thereof, the cryoprotectant is specially designed for testicular tissue suspension, the cryoprotectant can improve the cryoprotectant effect of testicular sperms, the problems of potential safety hazards, poor cryoprotectant effect, difficulty in finding moving testicular sperms and the like are effectively solved, and the cryoprotectant has very important clinical application value.
In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows: provides a human testicular tissue suspension cryoprotectant, wherein each 100mL of cryoprotectant comprises the following raw materials by weight: 0.35-0.65 g of sodium chloride, 0.03-0.045 g of potassium chloride, 0.035-0.047 g of calcium chloride, 0.008-0.0151 g of magnesium chloride, 0.004-0.007 g of monopotassium phosphate, 0.2-0.32 g of sodium bicarbonate, 0.3-0.55 g of HEPES, 0.035-0.058 g of caffeine, 0.0017-0.0035 g of sodium pyruvate, 0.102-0.555 g of pentoxifylline, 0.5-1.3 g of glycine, 0.08-0.209 g of glucose and 1-2.5 g of sucrose;
the human testicular tissue suspension cryoprotectant per 100mL further comprises 130-280 μ L of sodium lactate and 10-65 mL of glycerol.
Further, the human testicular tissue suspension cryoprotectant per 100mL comprises the following raw materials by weight: 0.584g of sodium chloride, 0.04g of potassium chloride, 0.04g of calcium chloride, 0.01g of magnesium chloride, 0.005g of monopotassium phosphate, 0.26g of sodium bicarbonate, 0.477g of HEPES, 0.048g of caffeine, 0.0028g of sodium pyruvate, 0.208g of pentoxifylline, 1g of glycine, 0.109g of glucose and 1.712g of sucrose.
Further, the cryoprotectant for every 100mL of human testicular tissue suspension comprises 188 μ L sodium lactate and 30mL glycerol.
The preparation method of the human testicular tissue suspension cryoprotectant comprises the following steps:
(1) sequentially weighing sodium chloride, potassium chloride, calcium chloride, magnesium chloride, potassium dihydrogen phosphate, sodium bicarbonate, HEPES, caffeine, sodium pyruvate, pentoxifylline, glycine, glucose and sucrose, and dissolving with ultrapure water to obtain a mixed solution;
(2) adding sodium lactate and glycerol into the mixed solution obtained in the step (1), adding ultrapure water to a constant volume, adjusting the pH value, sequentially filtering and carrying out bacterial culture, and subpackaging to obtain the human testicular tissue suspension cryoprotectant; the concentration of the sodium chloride after constant volume is 3.5-6.5 g/L.
Further, in the step (2), concentrated hydrochloric acid or 1mol/L sodium hydroxide solution is adopted to adjust the pH value to 7.36-7.4.
Further, the pore diameter of the filter membrane of the bacterial filter was 0.22 μm at the time of bacterial culture.
Further, the human testicular tissue suspension cryoprotectant prepared in step (2) is stored frozen at a temperature of-20 ℃ and below.
A method for freezing human testicular tissue suspension by using the human testicular tissue suspension cryoprotectant comprises the following steps:
placing human testis tissue in semen acceptor, cutting into tissue blocks without macroscopic view, adding human testis tissue suspension cryoprotectant, mixing, and placing in 37 deg.C incubator for 5 min; subpackaging, fumigating at 8cm position on liquid nitrogen for 10min, and preserving in liquid nitrogen; wherein the volume ratio of the semen to be washed to the human testicular tissue suspension cryoprotectant is 1: 1-4.
A method for resuscitating a frozen human testicular tissue suspension comprising the steps of:
taking out the frozen human testis tissue suspension from liquid nitrogen, heating in water bath at 37 deg.C for 1min, placing in sterile EP tube, and placing in 37 deg.C incubator for 15 min.
In summary, the invention has the following advantages:
1. the cryoprotectant is specially designed for the testicular tissue suspension, so that the cryopreservation effect of testicular sperms can be improved, the number of the movable sperms frozen by the testicular tissue suspension can be effectively improved, the movable sperms can be quickly found after recovery, the preparation method is convenient to operate, the cryoprotectant can be quickly prepared, the working efficiency is high, the problems of potential safety hazards, poor cryopreservation effect, difficulty in finding the movable testicular sperms and the like are effectively solved, the clinical utilization rate of the frozen testicular tissues after recovery is greatly improved, and the cryoprotectant has very important clinical application value.
2. The human testis tissue suspension cryoprotectant contains nonionic amphoteric buffer solution HEPES, can control the pH value within a constant range for a long time, and is added with glucose, sucrose, glycerol, glycine, pentoxifylline, caffeine and other substances. Sodium chloride, potassium chloride, calcium chloride, magnesium chloride, potassium dihydrogen phosphate, sodium bicarbonate, glycine, etc. as a balance liquid; wherein, the glucose and the sucrose are non-permeable cryoprotectants and can not enter cells, and the cryoprotectant has the main functions of maintaining the stability of cell membranes, and reducing the formation of ice crystals in cells by enabling water in the cells to overflow in the freezing process so as to achieve the protection effect. Glycerol is a permeable cryoprotectant, can enter cells, reduces the osmotic pressure difference inside and outside the cells, can reduce the freezing point of intracellular fluid, reduces the formation of ice crystals, plays a role in protecting sperm membranes and achieves the effect of cryoprotection. The caffeine and the pentoxifylline can effectively improve the proportion of moving sperms found after the testicular sperms are recovered. Different from the prior cryoprotectant, the cryoprotectant for the testicular tissue suspension does not contain exogenous substances such as yolk, albumin and the like, greatly improves the safety of specimen use, ensures clear vision after resuscitation and is beneficial to search sperms.
Detailed Description
Example 1
A human testicular tissue suspension cryoprotectant, each 100mL comprises the following raw materials by weight: 0.35g of sodium chloride, 0.03g of potassium chloride, 0.035g of calcium chloride, 0.008g of magnesium chloride, 0.004g of monopotassium phosphate, 0.2g of sodium bicarbonate, 0.3g of HEPES, 0.035g of caffeine, 0.0017g of sodium pyruvate, 0.102g of pentoxifylline, 0.5g of glycine, 0.08g of glucose and 1g of sucrose; also included are 130. mu.L of sodium lactate and 10mL of glycerol.
The preparation method of the human testicular tissue suspension cryoprotectant comprises the following steps:
(1) sequentially weighing sodium chloride, potassium chloride, calcium chloride, magnesium chloride, potassium dihydrogen phosphate, sodium bicarbonate, HEPES, caffeine, sodium pyruvate, pentoxifylline, glycine, glucose and sucrose, and dissolving with ultrapure water to obtain a mixed solution;
(2) adding sodium lactate and glycerol into the mixed solution obtained in the step (1), adding ultrapure water to a constant volume, adjusting the pH value to 7.36-7.4 by adopting concentrated hydrochloric acid or 1mol/L sodium hydroxide solution, then sequentially filtering and carrying out bacterial culture, subpackaging to obtain a human testicular tissue suspension cryoprotectant, and carrying out cryopreservation at the temperature of-20 ℃; the concentration of sodium chloride after constant volume is 3.5 g/L.
Example 2
A human testis tissue suspension cryoprotectant comprises the following raw materials by weight per 100mL of human testis tissue suspension: 0.584g of sodium chloride, 0.04g of potassium chloride, 0.04g of calcium chloride, 0.01g of magnesium chloride, 0.005g of monopotassium phosphate, 0.26g of sodium bicarbonate, 0.477g of HEPES, 0.048g of caffeine, 0.0028g of sodium pyruvate, 0.208g of pentoxifylline, 1g of glycine, 0.109g of glucose and 1.712g of sucrose; also comprises 188 mu L of sodium lactate and 30mL of glycerol.
The preparation method of the human testicular tissue suspension cryoprotectant comprises the following steps:
(1) sequentially weighing sodium chloride, potassium chloride, calcium chloride, magnesium chloride, potassium dihydrogen phosphate, sodium bicarbonate, HEPES, caffeine, sodium pyruvate, pentoxifylline, glycine, glucose and sucrose, and dissolving with ultrapure water to obtain a mixed solution;
(2) adding sodium lactate and glycerol into the mixed solution obtained in the step (1), adding ultrapure water to a constant volume, adjusting the pH value to 7.36-7.4 by adopting concentrated hydrochloric acid or 1mol/L sodium hydroxide solution, then sequentially filtering and carrying out bacterial culture, subpackaging to obtain a human testicular tissue suspension cryoprotectant, and carrying out cryopreservation at the temperature of-20 ℃; the concentration of the sodium chloride after constant volume is 5.84 g/L.
Example 3
A human testicular tissue suspension cryoprotectant, each 100mL comprises the following raw materials by weight: 0.65g of sodium chloride, 0.045g of potassium chloride, 0.047g of calcium chloride, 0.0151g of magnesium chloride, 0.007g of monopotassium phosphate, 0.32g of sodium bicarbonate, 0.55g of HEPES, 0.058g of caffeine, 0.0035g of sodium pyruvate, 0.555g of pentoxifylline, 1.3g of glycine, 0.209g of glucose and 2.5g of sucrose; also included are 280. mu.L of sodium lactate and 65mL of glycerol.
The preparation method of the human testicular tissue suspension cryoprotectant comprises the following steps:
(1) sequentially weighing sodium chloride, potassium chloride, calcium chloride, magnesium chloride, potassium dihydrogen phosphate, sodium bicarbonate, HEPES, caffeine, sodium pyruvate, pentoxifylline, glycine, glucose and sucrose, and dissolving with ultrapure water to obtain a mixed solution;
(2) adding sodium lactate and glycerol into the mixed solution obtained in the step (1), adding ultrapure water to a constant volume, adjusting the pH value to 7.36-7.4 by adopting concentrated hydrochloric acid or 1mol/L sodium hydroxide solution, then sequentially filtering and carrying out bacterial culture, subpackaging to obtain a human testicular tissue suspension cryoprotectant, and carrying out cryopreservation at the temperature of-20 ℃; the concentration of the sodium chloride after constant volume is 6.5 g/L.
Examples of the experiments
Experimental groups: 24 azoospermia patients are selected in an andrology clinic of a hospital, and after diagnostic testicular puncture and sperm finding, the human testicular tissue suspension cryoprotectant obtained in example 2 is adopted to carry out testicular tissue suspension freezing and resuscitation, the number of A, B, C, D-grade sperms in 5 mu L of human testicular tissue suspension before freezing and after resuscitation is counted respectively, and the result is shown in table 1.
Control group: meanwhile, 21 azoospermia patients are selected in the andrology clinic of a hospital, the patients with azoospermia are checked through diagnostic testicular puncture, then testis tissue suspension is frozen and revived by adopting a common cryoprotectant, the number of A, B, C, D-grade sperms in 5 mu L of human testis tissue suspension before freezing and after reviving is counted respectively, and the result is shown in table 2. After the experimental group and the control group are recovered, the proportion of the moving sperms is checked and analyzed in a comparison mode, and the result is shown in table 3.
The human testicular tissue suspension freezing method used in the experimental group and the control group comprises the following steps: placing human testis tissue in 300 μ L semen washing receiving fluid, cutting into tissue blocks without macroscopic view, adding the human testis tissue suspension cryoprotectant at volume ratio of 1:3, mixing, and placing in incubator at 37 deg.C for 5 min; counting 5 mu L of the testis tissue suspension, and respectively recording the number of A, B, C, D-grade sperms; subpackaging, fumigating at 8cm position on liquid nitrogen for 10min, and storing in liquid nitrogen.
The recovery method of the frozen human testicular tissue suspension adopted in the experimental group and the control group comprises the following steps: taking the frozen human testis tissue suspension out of liquid nitrogen, heating in water bath at 37 deg.C for 1min, placing in sterile EP tube, placing in incubator at 37 deg.C for 15min, and recording A, B, C, D grade sperm number.
TABLE 124 patient testicular tissue pre-freezing and post-resuscitation results
TABLE 221 patient testicular tissue pre-freezing and post-resuscitation results
TABLE 3 comparison of ratio of sperm to sperm after resuscitation in experimental and control groups
As can be seen from tables 1-3, 24 cases of patients in the experimental group had testis tissue suspensions frozen and preserved by the testis tissue suspension cryoprotectant obtained by the invention, and after resuscitation, the active testis sperms were observed, and the proportion was 100%; the testis tissue suspension of 21 patients in the control group is frozen and preserved by adopting a common cryoprotectant, and the number of the movable sperms is 9 after recovery, and the proportion is 42.85%. And the P value of the two compared is less than 0.05, and the difference has obvious statistical significance.
The results show that the testis tissue suspension cryoprotectant provided by the invention has better freezing effect than the common cryoprotectant, can better protect testis sperms and improve the testis sperm freezing recovery effect.
While the present invention has been described in detail with reference to the specific embodiments thereof, it should not be construed as limited by the scope of the present patent. Various modifications and changes may be made by those skilled in the art without inventive step within the scope of the appended claims.
Claims (8)
1. A human testicular tissue suspension cryoprotectant is characterized in that each 100mL of cryoprotectant comprises the following raw materials by weight: 0.35-0.65 g of sodium chloride, 0.03-0.045 g of potassium chloride, 0.035-0.047 g of calcium chloride, 0.008-0.0151 g of magnesium chloride, 0.004-0.007 g of monopotassium phosphate, 0.2-0.32 g of sodium bicarbonate, 0.3-0.55 g of HEPES, 0.035-0.058 g of caffeine, 0.0017-0.0035 g of sodium pyruvate, 0.102-0.555 g of pentoxifylline, 0.5-1.3 g of glycine, 0.08-0.209 g of glucose and 1-2.5 g of sucrose;
the human testicular tissue suspension cryoprotectant per 100mL further comprises 130-280 μ L of sodium lactate and 10-65 mL of glycerol.
2. The human testicular tissue suspension cryoprotectant of claim 1, wherein the human testicular tissue suspension cryoprotectant per 100mL comprises the following raw materials by weight: 0.584g of sodium chloride, 0.04g of potassium chloride, 0.04g of calcium chloride, 0.01g of magnesium chloride, 0.005g of monopotassium phosphate, 0.26g of sodium bicarbonate, 0.477g of HEPES, 0.048g of caffeine, 0.0028g of sodium pyruvate, 0.208g of pentoxifylline, 1g of glycine, 0.109g of glucose and 1.712g of sucrose.
3. The human testicular tissue suspension cryoprotectant of claim 1, wherein said cryoprotectant comprises 188 μ L of said sodium lactate and 30mL of said glycerol per 100mL of said human testicular tissue suspension.
4. A method of preparing a human testicular tissue suspension cryoprotectant of any of claims 1-3, comprising the steps of:
(1) sequentially weighing sodium chloride, potassium chloride, calcium chloride, magnesium chloride, potassium dihydrogen phosphate, sodium bicarbonate, HEPES, caffeine, sodium pyruvate, pentoxifylline, glycine, glucose and sucrose, and dissolving with ultrapure water to obtain a mixed solution;
(2) adding sodium lactate and glycerol into the mixed solution obtained in the step (1), adding ultrapure water to a constant volume, adjusting the pH value, sequentially filtering and carrying out bacterial culture, and subpackaging to obtain the human testicular tissue suspension cryoprotectant; the concentration of the sodium chloride after constant volume is 3.5-6.5 g/L.
5. The method according to claim 1, wherein in step (2), concentrated hydrochloric acid or 1mol/L NaOH solution is used to adjust the pH value to 7.36-7.4.
6. The method of claim 1 wherein the bacterial culture is performed with a bacterial filter membrane having a pore size of 0.22 μm.
7. The method of claim 1, wherein the human testicular tissue suspension cryoprotectant prepared in step (2) is stored frozen at a temperature of-20 ℃ or below.
8. A method of freezing a human testicular tissue suspension using the human testicular tissue suspension cryoprotectant of any one of claims 1-3, comprising the steps of:
placing human testis tissue in semen acceptor, cutting into tissue blocks without macroscopic view, adding human testis tissue suspension cryoprotectant, mixing, and placing in 37 deg.C incubator for 5 min; subpackaging, fumigating at 8cm position on liquid nitrogen for 10min, and preserving in liquid nitrogen; wherein the volume ratio of the semen-washed and semen-receiving liquid to the human testicular tissue suspension cryoprotectant is 1: 1-4.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112273369A (en) * | 2020-09-21 | 2021-01-29 | 上海市第十人民医院 | Sperm cryopreservation liquid for maintaining sperm plasma membrane stability and preparation method and application thereof |
| CN113005077A (en) * | 2021-03-19 | 2021-06-22 | 吉林大学第一医院 | Preparation method of in-vitro testicular tissue living single sperm suspension |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103999849A (en) * | 2014-05-07 | 2014-08-27 | 西北农林科技大学 | Antifreezing agent for mammal testis tissue freezing preservation and freezing-unfreezing method |
| CN104046590A (en) * | 2014-06-18 | 2014-09-17 | 郜鸿生物科技（上海）有限公司 | In-vitro culture medium for sperm from oligospermia and asthenospermia patients |
| CN104365583A (en) * | 2014-11-07 | 2015-02-25 | 郜鸿生物科技（上海）有限公司 | Extremely-low sperm freezing protective agent and application thereof |
| CN106818707A (en) * | 2017-01-16 | 2017-06-13 | 上海尚维生物科技有限公司 | Pet testis tissue cryopreservation resuscitation reagent and cryopreservation resuscitation method |
| US20190174742A1 (en) * | 2016-08-22 | 2019-06-13 | Bioverde Inc. | Composition for cryopreservation of bovine reproductive cells and cryopreservation method thereof |
-
2020
- 2020-04-09 CN CN202010276178.4A patent/CN111345284B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103999849A (en) * | 2014-05-07 | 2014-08-27 | 西北农林科技大学 | Antifreezing agent for mammal testis tissue freezing preservation and freezing-unfreezing method |
| CN104046590A (en) * | 2014-06-18 | 2014-09-17 | 郜鸿生物科技（上海）有限公司 | In-vitro culture medium for sperm from oligospermia and asthenospermia patients |
| CN104365583A (en) * | 2014-11-07 | 2015-02-25 | 郜鸿生物科技（上海）有限公司 | Extremely-low sperm freezing protective agent and application thereof |
| US20190174742A1 (en) * | 2016-08-22 | 2019-06-13 | Bioverde Inc. | Composition for cryopreservation of bovine reproductive cells and cryopreservation method thereof |
| CN106818707A (en) * | 2017-01-16 | 2017-06-13 | 上海尚维生物科技有限公司 | Pet testis tissue cryopreservation resuscitation reagent and cryopreservation resuscitation method |
Non-Patent Citations (3)
| Title |
|---|
| 吕家驹等: "《实用临床男科学》", 31 December 2005, 山东大学出版社 * |
| 朱伟杰等: "人类精液一步冷冻法的研究", 《中国病理生理杂志》 * |
| 李济宇等: "《医疗机构知识产权保护案例精选》", 31 December 2018, 上海科学技术出版社 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112273369A (en) * | 2020-09-21 | 2021-01-29 | 上海市第十人民医院 | Sperm cryopreservation liquid for maintaining sperm plasma membrane stability and preparation method and application thereof |
| CN113005077A (en) * | 2021-03-19 | 2021-06-22 | 吉林大学第一医院 | Preparation method of in-vitro testicular tissue living single sperm suspension |
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