CN103275930A - 一种卵母细胞体外成熟培养液及其制备方法 - Google Patents
一种卵母细胞体外成熟培养液及其制备方法 Download PDFInfo
- Publication number
- CN103275930A CN103275930A CN201310247051XA CN201310247051A CN103275930A CN 103275930 A CN103275930 A CN 103275930A CN 201310247051X A CN201310247051X A CN 201310247051XA CN 201310247051 A CN201310247051 A CN 201310247051A CN 103275930 A CN103275930 A CN 103275930A
- Authority
- CN
- China
- Prior art keywords
- oocyte
- nutrient solution
- vitro
- human
- commercialized
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 210000000287 oocyte Anatomy 0.000 title claims abstract description 26
- 238000000338 in vitro Methods 0.000 title claims abstract description 21
- 235000015097 nutrients Nutrition 0.000 title claims abstract description 20
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- 210000002966 serum Anatomy 0.000 claims abstract description 11
- 102000008100 Human Serum Albumin Human genes 0.000 claims abstract description 8
- 108091006905 Human Serum Albumin Proteins 0.000 claims abstract description 8
- 230000035800 maturation Effects 0.000 claims description 21
- 210000004027 cell Anatomy 0.000 claims description 9
- 150000000307 17β-estradiols Chemical class 0.000 claims description 6
- 238000005138 cryopreservation Methods 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- VOXZDWNPVJITMN-ZBRFXRBCSA-N 17β-estradiol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 VOXZDWNPVJITMN-ZBRFXRBCSA-N 0.000 abstract description 6
- 102000007562 Serum Albumin Human genes 0.000 abstract description 5
- 108010071390 Serum Albumin Proteins 0.000 abstract description 5
- 229960005309 estradiol Drugs 0.000 abstract description 4
- 230000035935 pregnancy Effects 0.000 abstract description 4
- 230000007012 clinical effect Effects 0.000 abstract description 2
- 229930182833 estradiol Natural products 0.000 abstract description 2
- 238000001727 in vivo Methods 0.000 abstract description 2
- 239000000470 constituent Substances 0.000 abstract 2
- 239000001963 growth medium Substances 0.000 abstract 2
- 239000000243 solution Substances 0.000 description 25
- 238000002474 experimental method Methods 0.000 description 5
- 230000019100 sperm motility Effects 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- 102000002322 Egg Proteins Human genes 0.000 description 3
- 108010000912 Egg Proteins Proteins 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 230000008175 fetal development Effects 0.000 description 3
- 210000001161 mammalian embryo Anatomy 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 210000004681 ovum Anatomy 0.000 description 3
- 239000002574 poison Substances 0.000 description 3
- 231100000614 poison Toxicity 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 230000034004 oogenesis Effects 0.000 description 2
- 210000003101 oviduct Anatomy 0.000 description 2
- 210000005132 reproductive cell Anatomy 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 210000000582 semen Anatomy 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 206010013183 Dislocation of vertebra Diseases 0.000 description 1
- 206010036590 Premature baby Diseases 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 230000003187 abdominal effect Effects 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000007599 discharging Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000004720 fertilization Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 210000002503 granulosa cell Anatomy 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 210000004508 polar body Anatomy 0.000 description 1
- 201000010065 polycystic ovary syndrome Diseases 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 239000003104 tissue culture media Substances 0.000 description 1
- 230000036228 toxication Effects 0.000 description 1
- 239000006216 vaginal suppository Substances 0.000 description 1
- 229940120293 vaginal suppository Drugs 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明属于辅助生殖技术领域，具体涉及一种卵母细胞体外成熟培养液及其制备方法。本发明所述卵母细胞体外成熟培养液由商品化的组织培养液TCM199为基础，精确添加明确的已经商品化的成分包括0.075-0.10IU/ml FSH和0.5-0.8IU/ml HCG，0.1-0.2μg/ml的17β-雌二醇，和10%-20%体积百分比的人商品化替代血清或者人白蛋白。本发明所述卵母细胞体外成熟培养液成分稳定，质量稳定，可以产业化生产。通过添加商品化的人替代血清，血清白蛋白以及提高培养液中雌二醇，FSH，HCG的浓度，模拟人卵母细胞体内成熟的环境获得了很好的临床效果，卵母细胞体外成熟率达到了72.08%，临床妊娠率达到了23.71%。
Description
技术领域
本发明属于辅助生殖技术领域，具体涉及一种卵母细胞体外成熟培养液及其制备方法。
背景技术
现有的用于培养女性生殖细胞在体外快速成熟的培养液中大多添加胎牛血清或者人成熟卵泡液，容易导致异种病原物的传播以及同种间不明原因疾病的散播，易导致新生儿的缺陷，不利于优生优育。另外各种添加物的细化成分不明确，导致临床结果不稳定以及无法产业化。
发明内容
本发明的目的是提供一种成分稳定，质量稳定，可以产业化生产的卵母细胞体外成熟培养液及其制备方法，以解决现有技术中存在的由于各种添加物的细化成分不明确，导致临床结果不稳定以及无法产业化问题。
本发明所采用的技术方案是：
本发明所述卵母细胞体外成熟培养液是以商品化的组织培养液TCM199（Sigma,St.Louis.USA）为基础，精确添加明确的已经商品化的成分包括0.075-0.10IU/ml FSH和0.5-0.8IU/ml HCG（Serono公司，瑞士），0.1-0.2μg/ml的17β-雌二醇（Sigma,St.Louis,USA），和10%-20%体积百分比的人商品化替代血清或者人白蛋白。
本发明所述卵母细胞体外成熟培养液的制备方法是将基本细胞培养液TCM199添加0.075-0.10IU/ml FSH，0.5-0.8IU/ml HCG，用0.22μm的过滤器过滤，4℃低温保存，3周内使用。使用前再依次添加0.1-0.2μg/ml的17β-雌二醇，10%-20%的人商品化替代血清或者人白蛋白，充分混匀。培养卵母细胞前培养液于37℃培养箱，5%二氧化碳浓度条件下平衡12小时以上。
TCM199是一种经典的细胞体外培养系统，用其作为母液安全、可靠。
FSH，HCG以及17β-雌二醇为人体内卵母细胞成熟的必须激素，其含量是依据其体内成熟环境以及多年的临床试验数据设定。
人商品化替代血清或人白蛋白与体内组织液环境类似，商品组分稳定安全。
FSH，HCG以及17β-雌二醇主要是促进未成熟卵母细胞周围颗粒细胞松散，促进未成熟卵母细胞核成熟排出极体。与人商品化替代血清或人白蛋白合用能进一步促进细胞质的成熟。为人卵母细胞成熟后的受精及形成胚胎后的体外发育以及临床妊娠打下坚实的基础。
本发明的有益效果是：
本发明所述卵母细胞体外成熟培养液，培养女性生殖细胞可以在体外快速成熟，该培养液成分稳定，质量稳定，可以产业化生产。通过添加商品化的人替代血清，血清白蛋白以及提高培养液中雌二醇，FSH，HCG的浓度，模拟人卵母细胞体内成熟的环境获得了很好的临床效果，卵母细胞体外成熟率达到了72.08%，临床妊娠率达到了23.71%。
具体实施方式
1.卵母细胞体外成熟培养液的制备
本发明所述卵母细胞体外成熟培养液的制备方法：
将基本细胞培养液TCM199添加0.075-0.10IU/ml FSH，0.5-0.8IU/mlHCG，用0.22μm的过滤器过滤，4℃低温保存，3周内使用。使用前再依次添加0.1-0.2μg/ml的17β-雌二醇，10%-20%的人商品化替代血清或者人白蛋白，充分混匀。培养卵母细胞前培养液于37℃培养箱，5%二氧化碳浓度条件下平衡12小时以上。
添加和配置要求在GMP车间环境下进行，要求无菌，无毒，无尘。
2.卵母细胞体外成熟培养液的急毒和长毒试验
采用人精子存活实验，小鼠胚胎培养实验进行急毒和长毒实验。
人精子存活实验：
使用正常水平的精液标本，利用上游法分离活动精子。分两管，分别加入待检测的培养液（T）和对照培养液（C），混匀并调整精子密度至5×106／ml。取混匀后的精子悬液用Markler精子计数板做精液常规检查，测定两组的精子活率。将精子悬液置于室温或CO2培养箱中孵育，分别于24小时、48小时、72小时和96小时后测定两组精子活率。计算生存指数（活率指数）＝T组精子活率/C组精子活率，生存指数＞75%为合格。
鼠胚实验：
选择4～7周龄雌性小鼠，每只腹腔注射10IU PMSG，48小时后注射10IUhCG。当晚按雌雄比例3:1合笼。第二日观察阴道栓，阳性者记为第一天。第三天颈椎脱臼法处死雌鼠，撕开腹部皮肤，打开腹腔。更换剪刀和镊子，剪下输卵管置于培养液中。在解剖显微镜下撕开输卵管壶腹部，取出受精卵，充分洗涤后移入待检测的培养液（T）和对照培养液（C），CO2培养箱中孵育。72小时后观察囊胚形成率，2细胞胚胎形成囊胚的比例超过70%视为合格。
3.卵母细胞体外成熟培养液的临床观察
应用自行研配的成熟培养液系统，进行未成熟卵母细胞在体外成熟培养的研究和临床运用。收集5例行小卵泡穿刺的PCOS患者的未成熟卵母细胞作为试验对象，进行体外成熟、胚胎发育的研究。
表1.培养液对小卵泡穿刺术卵子成熟及胚胎发育的影响
表2.培养液对卵子成熟及胚胎发育的结果
表3.培养液培养的卵子的胚胎种植及临床妊娠的能力
Claims (2)
1.一种卵母细胞体外成熟培养液，其特征是由基本细胞培养液TCM199添加0.075-0.10IU/ml FSH，0.5-0.8IU/ml HCG，0.1-0.2μg/ml的17β-雌二醇，10%-20%体积百分比的人商品化替代血清或者人白蛋白组成。
2.根据权利要求1所述卵母细胞体外成熟培养液的制备方法，其特征是将基本细胞培养液TCM199添加0.075-0.10IU/ml FSH，0.5-0.8IU/ml HCG，用0.22μm的过滤器过滤，4℃低温保存，3周内使用；使用前再依次添加0.1-0.2μg/ml的17β-雌二醇，10%-20%体积百分比的人商品化替代血清或者人白蛋白，充分混匀；培养卵母细胞前培养液于37℃培养箱，5%二氧化碳浓度条件下平衡12小时以上。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310247051.XA CN103275930B (zh) | 2013-06-20 | 2013-06-20 | 一种卵母细胞体外成熟培养液及其制备方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310247051.XA CN103275930B (zh) | 2013-06-20 | 2013-06-20 | 一种卵母细胞体外成熟培养液及其制备方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103275930A true CN103275930A (zh) | 2013-09-04 |
| CN103275930B CN103275930B (zh) | 2014-07-09 |
Family
ID=49058554
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310247051.XA Expired - Fee Related CN103275930B (zh) | 2013-06-20 | 2013-06-20 | 一种卵母细胞体外成熟培养液及其制备方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103275930B (zh) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104140952A (zh) * | 2014-08-08 | 2014-11-12 | 山东威高新生医疗器械有限公司 | 透明质酸酶及其制备方法 |
| CN105518124A (zh) * | 2015-06-26 | 2016-04-20 | 深圳市第二人民医院 | 一种猪卵母细胞体外成熟培养液及其制备方法和培养方法 |
| CN107058215A (zh) * | 2017-01-20 | 2017-08-18 | 江苏省中医院 | 一种添加中药单体的猪卵母细胞体外受精液及培养方法 |
| CN108823152A (zh) * | 2018-07-25 | 2018-11-16 | 河南牧业经济学院 | 一种小鼠卵母细胞的体外成熟培养液及体外成熟培养方法 |
| CN108949670A (zh) * | 2018-08-16 | 2018-12-07 | 中国人民解放军第四军医大学 | 一种卵母细胞体外成熟培养液的制备方法 |
| CN109628386A (zh) * | 2019-01-18 | 2019-04-16 | 周桦 | 一种人卵母细胞的体外成熟培养液制备方法和培养方法 |
| CN115029303A (zh) * | 2022-06-22 | 2022-09-09 | 安徽第肆纪医学科技有限公司 | 一种用于coh周期中人未成熟卵母细胞ivm能量培养液 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1226377A (zh) * | 1999-04-01 | 1999-08-25 | 旭日干 | “试管牛”技术产业化工艺流程 |
| CN1226378A (zh) * | 1999-04-01 | 1999-08-25 | 旭日干 | 羊“试管内”杂交育种技术 |
| WO1999067644A1 (en) * | 1998-06-22 | 1999-12-29 | Medi-Cult A/S | Assay to indicate the presence of non-fertilisable ova |
-
2013
- 2013-06-20 CN CN201310247051.XA patent/CN103275930B/zh not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999067644A1 (en) * | 1998-06-22 | 1999-12-29 | Medi-Cult A/S | Assay to indicate the presence of non-fertilisable ova |
| CN1313951A (zh) * | 1998-06-22 | 2001-09-19 | 梅迪-卡尔特公司 | 指示未受精卵存在的测定法 |
| CN1226377A (zh) * | 1999-04-01 | 1999-08-25 | 旭日干 | “试管牛”技术产业化工艺流程 |
| CN1226378A (zh) * | 1999-04-01 | 1999-08-25 | 旭日干 | 羊“试管内”杂交育种技术 |
Non-Patent Citations (1)
| Title |
|---|
| 马红等: "激素对猪卵母细胞体外成熟及孤雌胚胎发育的影响", 《畜牧与兽医》, vol. 45, no. 4, 30 April 2013 (2013-04-30), pages 41 - 43 * |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104140952A (zh) * | 2014-08-08 | 2014-11-12 | 山东威高新生医疗器械有限公司 | 透明质酸酶及其制备方法 |
| CN104140952B (zh) * | 2014-08-08 | 2018-03-16 | 山东威高新生医疗器械有限公司 | 透明质酸酶及其制备方法 |
| CN105518124A (zh) * | 2015-06-26 | 2016-04-20 | 深圳市第二人民医院 | 一种猪卵母细胞体外成熟培养液及其制备方法和培养方法 |
| WO2016206086A1 (zh) * | 2015-06-26 | 2016-12-29 | 深圳市第二人民医院 | 一种猪卵母细胞体外成熟培养液及其制备方法和培养方法 |
| CN107058215A (zh) * | 2017-01-20 | 2017-08-18 | 江苏省中医院 | 一种添加中药单体的猪卵母细胞体外受精液及培养方法 |
| CN108823152A (zh) * | 2018-07-25 | 2018-11-16 | 河南牧业经济学院 | 一种小鼠卵母细胞的体外成熟培养液及体外成熟培养方法 |
| CN108949670A (zh) * | 2018-08-16 | 2018-12-07 | 中国人民解放军第四军医大学 | 一种卵母细胞体外成熟培养液的制备方法 |
| CN109628386A (zh) * | 2019-01-18 | 2019-04-16 | 周桦 | 一种人卵母细胞的体外成熟培养液制备方法和培养方法 |
| CN115029303A (zh) * | 2022-06-22 | 2022-09-09 | 安徽第肆纪医学科技有限公司 | 一种用于coh周期中人未成熟卵母细胞ivm能量培养液 |
| CN115029303B (zh) * | 2022-06-22 | 2023-08-15 | 安徽第肆纪医学科技有限公司 | 一种用于coh周期中人未成熟卵母细胞ivm能量培养液 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103275930B (zh) | 2014-07-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103275930B (zh) | 一种卵母细胞体外成熟培养液及其制备方法 | |
| Hall | Relationship between semen quality and human sperm penetration of zona-free hamster ova | |
| Fry et al. | Human leukemia inhibitory factor improves the viability of cultured ovine embryos | |
| Agarwal et al. | Oxidative stress and its implications in female infertility–a clinician's perspective | |
| Kasterstein et al. | The effect of two distinct levels of oxygen concentration on embryo development in a sibling oocyte study | |
| Zhao et al. | In vitro fertilization: four decades of reflections and promises | |
| MacLennan et al. | THE ULTRASTRUCTURE OF HUMAN TROPHOBLAST IN SPONTANEOUS AND INDUCED HYPOXIA USING A SYSTEM OF ORGAN CULTURE: A Comparison with Ultrastructural Changes in Pre‐eclampsia and Placental Insufficiency | |
| Lueders et al. | Gestating for 22 months: luteal development and pregnancy maintenance in elephants | |
| Yániz et al. | Effects of solid storage of sheep spermatozoa at 15 C on their survival and penetrating capacity | |
| Huang et al. | Birth after the injection of sperm and the cytoplasm of tripronucleate zygotes into metaphase II oocytes in patients with repeated implantation failure after assisted fertilization procedures | |
| Marco-Jiménez et al. | Influence of zona pellucida thickness on fertilization, embryo implantation and birth | |
| Balaban et al. | Laboratory procedures for human in vitro fertilization | |
| García-Martínez et al. | Mimicking the temperature gradient between the sow’s oviduct and uterus improves in vitro embryo culture output | |
| Jin et al. | The value of second polar body detection 4 hours after insemination and early rescue ICSI in preventing complete fertilisation failure in patients with borderline semen | |
| Veres et al. | The biology and methodology of assisted reproduction in deer mice (Peromyscus maniculatus) | |
| CN103333855B (zh) | 一种羊胚胎细胞培养液 | |
| Dai et al. | Effect of coincubation time of sperm-oocytes on fertilization, embryonic development, and subsequent pregnancy outcome | |
| CN107460162B (zh) | 一种提高羔羊体外胚胎发育能力的方法 | |
| Morton et al. | In vitro and in vivo developmental capabilities and kinetics of in vitro development of in vitro matured oocytes from adult, unstimulated and hormone-stimulated prepubertal ewes | |
| Caillaud et al. | In vitro equine oocyte maturation in pure follicular fluid plus interleukin-1 and fertilization following ICSI | |
| Bhattacharya et al. | Regenerative medicine using pregnancy-specific biological substances | |
| Jixiang et al. | Relationship of sperm motility with clinical outcome of percutaneous epididymal sperm aspiration–intracytoplasmic sperm injection in infertile males with congenital domestic absence of vas deferens: a retrospective study | |
| Cossiello et al. | Morphological differences in human zygotes and embryos cultured in different media | |
| CN102174467A (zh) | 一种小鼠卵巢组织液的提取方法及其应用 | |
| Peters et al. | Culture and transfer of sheep embryos |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140709 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |