CN108753690A - 一种一步培养液及其制备方法 - Google Patents
一种一步培养液及其制备方法 Download PDFInfo
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- CN108753690A CN108753690A CN201810610841.2A CN201810610841A CN108753690A CN 108753690 A CN108753690 A CN 108753690A CN 201810610841 A CN201810610841 A CN 201810610841A CN 108753690 A CN108753690 A CN 108753690A
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Abstract
本发明涉及一种一步培养液及其制备方法,属于人类辅助生殖技术领域。所述一步培养液是向盐溶液内添加丙氨酸、天冬氨酸、天冬酰胺、谷氨酸、谷氨酰胺、甘氨酸、脯氨酸、牛磺酸、丝氨酸、苏氨酸、酪氨酸、甲硫氨酸、硫辛酸、透明质酸、精氨酸、胱氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、苯丙氨酸、苏氨酸、色氨酸、缬氨酸、叶酸、肌醇、维生素B12、维生素B6、维生素B5、维生素B2、维生素B1、硫胺素、次黄嘌呤、泛酸和螯合剂,不仅能有效保持胚胎的活力和营养,而且,螯合剂可以螯合一些非必要的重金属离子,降低培养液的毒性,有利于促进早期胚胎的发育和囊胚形成,提高辅助生殖技术的成功率,也为辅助生殖技术的安全性提供了有力保证。
Description
技术领域
本发明涉及一种一步培养液及其制备方法,属于人类辅助生殖技术领域。
背景技术
人类辅助生殖技术中,一步培养液是主要用于培养第1天至第5/6天的人胚胎和移植流程。目前,临床使用的常规的一步培养液为仅以碳酸氢钠为缓冲剂的序贯培养液,不能有效地确保胚胎的同步发育和旁分泌或者自分泌影响,尤其对早期胚胎的囊胚发育效果欠佳。
发明内容
本发明的目的在于,提供一种各项质控达标的一步培养液,不仅能有效地保持胚胎的活力,提高辅助生殖技术中早期胚胎的发育率和囊胚形成率,也为辅助生殖技术的安全性提供有力保证。
为解决上述问题,本发明所采用的技术方案是:
一种一步培养液,其特征在于:是将丙氨酸、天冬氨酸、天冬酰胺、谷氨酸、谷氨酰胺、甘氨酸、脯氨酸、牛磺酸、丝氨酸、苏氨酸、酪氨酸、甲硫氨酸、硫辛酸、透明质酸、精氨酸、胱氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、苯丙氨酸、苏氨酸、色氨酸、缬氨酸、叶酸、肌醇、维生素B12、维生素B6、维生素B5、维生素B2、维生素B1、硫胺素、次黄嘌呤、泛酸和螯合剂溶解于含有抗菌素和指示剂的盐溶液中配制而成;
所述一步培养液中,丙氨酸、天冬氨酸、天冬酰胺、谷氨酸、谷氨酰胺、甘氨酸、脯氨酸、牛磺酸、丝氨酸、苏氨酸、酪氨酸、甲硫氨酸、硫辛酸、精氨酸、胱氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、苯丙氨酸、苏氨酸、色氨酸、缬氨酸的浓度均为0.01-1.00mmol/L;透明质酸的浓度为0.01-0.5mg/mL;叶酸、肌醇、维生素B12、维生素B6、维生素B5、维生素B2、维生素B1、硫胺素、次黄嘌呤、泛酸的浓度均为0.001-0.015mmol/L;螯合剂适量。
优选的:所述的螯合剂为四元羧酸乙二胺四乙酸。
优选的:所述抗菌素为庆大霉素,浓度为4-12μg/mL。
优选的:所述指示剂为酚红,浓度4.5-10.0μg/mL。
优选的:所述盐溶液是以碳酸氢钠为缓冲剂,以钠、钾、镁、钙离子为基础,并以葡萄糖、丙酮酸钠、乳酸钠、白蛋白为能量物质的化合物溶液;其中包含:氯化钠89.00-108.00mmol/L、氯化钾4.00-6.00mmol/L、硫酸镁0.10-1.20mmol/L、磷酸二氢钾0.3-0.6mmol/L、磷酸二氢钠0.10-0.50mmol/L、氯化钙1.60-2.50mmol/L、碳酸氢钠18.00-26.00mmol/L、葡萄糖0.30-0.80mmol/L、丙酮酸钠0.20-0.60mmol/L、乳酸钠9.00-23.00mmol/L、磷酸二氢钾0.20-0.50mmol/L;
所述白蛋白为重组人血清白蛋白,是采用医疗级的重组人血清白蛋白干粉溶解于盐水溶液中制备而成,终浓度为0.0-6.0mg/mL。
本发明所述一步培养液的制备方法,包括以下步骤:
1、配制盐溶液:先按照盐溶液的各组分用量称量好各种组分,备用;再将除了抗菌素、指示剂、碳酸氢钠以外的各组分溶于超纯注射级用水中,溶解过程中遵循先固体后液体的原则;所述超纯注射级用水经0.1-0.2μM滤膜过滤,内毒素<0.015EU/mL;然后,依次加入称量好的抗菌素、指示剂、碳酸氢钠,制得盐溶液;
2、检测步骤1所得盐溶液的渗透压和pH值,并记录最终渗透压和pH值;所述渗透压保持为260-285mOsm/Kg,所述pH值保持为7.30-7.80;
3、按照预配制的容量,将重组人血清白蛋白按终浓度为0.0-6.0mg/mL补充到盐溶液中;
4、将丙氨酸、天冬氨酸、天冬酰胺、谷氨酸、谷氨酰胺、甘氨酸、脯氨酸、牛磺酸、丝氨酸、苏氨酸、酪氨酸、甲硫氨酸、硫辛酸、精氨酸、胱氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、苯丙氨酸、苏氨酸、色氨酸、缬氨酸分别以0.01-1.00mmol/L的浓度溶解于盐溶液中;将透明质酸按照0.01-0.50mg/mL的浓度溶解于盐溶液中;将叶酸、肌醇、维生素B12、维生素B6、维生素B5、维生素B2、维生素B1、硫胺素、次黄嘌呤、泛酸分别以0.001-0.020mmol/L的浓度溶解于盐溶液中;将适量螯合剂溶解于盐溶液中;到充分溶解止;
5、将步骤4所得溶液经过0.2μm滤膜过滤灭菌后,取样测试;测试参数:
A、20-30℃条件下pH值:7.30-7.80;
B、渗透压:260-285mOsm/Kg;
C、内毒素:<0.15EU/mL;
D、一细胞鼠胚培养到96小时:≥80%的囊胚形成率;
6、将配制好的溶液在百级GMP车间进行无菌分装和标签。
有益效果:本发明所述的一步培养液是向盐溶液内添加丙氨酸、天冬氨酸、天冬酰胺、谷氨酸、谷氨酰胺、甘氨酸、脯氨酸、牛磺酸、丝氨酸、苏氨酸、酪氨酸、甲硫氨酸、硫辛酸、透明质酸、精氨酸、胱氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、苯丙氨酸、苏氨酸、色氨酸、缬氨酸、叶酸、肌醇、维生素B12、维生素B6、维生素B5、维生素B2、维生素B1、硫胺素、次黄嘌呤、泛酸和螯合剂,不仅可以有效地保持胚胎的活力和营养,而且,螯合剂还可以螯合一些非必要的重金属离子,降低培养液的毒性,有利于促进早期胚胎的发育和囊胚形成,提高辅助生殖技术的成功率,也为辅助生殖技术的安全性提供了有力保证。
具体实施方式
下面结合具体实施实施方式对本发明做进一步说明。
本发明所述的一步培养液,是将丙氨酸、天冬氨酸、天冬酰胺、谷氨酸、谷氨酰胺、甘氨酸、脯氨酸、牛磺酸、丝氨酸、苏氨酸、酪氨酸、甲硫氨酸、硫辛酸、透明质酸、精氨酸、胱氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、苯丙氨酸、苏氨酸、色氨酸、缬氨酸、叶酸、肌醇、维生素B12、维生素B6、维生素B5、维生素B2、维生素B1、硫胺素、次黄嘌呤、泛酸和螯合剂溶解于含有抗菌素和指示剂的盐溶液中配制而成。
所述一步培养液中,丙氨酸、天冬氨酸、天冬酰胺、谷氨酸、谷氨酰胺、甘氨酸、脯氨酸、牛磺酸、丝氨酸、苏氨酸、酪氨酸、甲硫氨酸、硫辛酸、精氨酸、胱氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、苯丙氨酸、苏氨酸、色氨酸、缬氨酸的浓度均为0.01-1.00mmol/L;透明质酸的浓度为0.01-0.50mg/mL;叶酸、肌醇、维生素B12、维生素B6、维生素B5、维生素B2、维生素B1、硫胺素、次黄嘌呤、泛酸的浓度均为0.001-0.020mmol/L;螯合剂适量。
所述的螯合剂为四元羧酸乙二胺四乙酸;所述抗菌素为庆大霉素,浓度为4-12μg/mL;所述指示剂为酚红,浓度4.5-10.0μg/mL。
所述盐溶液是以碳酸氢钠为缓冲剂,以钠、钾、镁、钙离子为基础,并以葡萄糖、丙酮酸钠、乳酸钠、白蛋白为能量物质的化合物溶液;其中包含:氯化钠89.00-108.00mmol/L、氯化钾4.00-6.00mmol/L、硫酸镁0.10-1.20mmol/L、磷酸二氢钾0.3-0.6mmol/L、磷酸二氢钠0.10-0.50mmol/L、氯化钙1.60-2.50mmol/L、碳酸氢钠18.00-26.00mmol/L、葡萄糖0.30-0.80mmol/L、丙酮酸钠0.20-0.60mmol/L、乳酸钠9.00-23.00mmol/L、磷酸二氢钾0.20-0.50mmol/L;所述白蛋白为重组人血清白蛋白,是采用医疗级的重组人血清白蛋白干粉溶解于盐水溶液中制备而成,终浓度为0.0-6.0mg/mL。
本发明所述一步培养液的制备方法,包括以下步骤:
1、配制盐溶液:先按照盐溶液的各组分用量称量好各种组分,备用;再将除了抗菌素、指示剂、碳酸氢钠以外的各组分溶于超纯注射级用水中,溶解过程中遵循先固体后液体的原则;所述超纯注射级用水经0.1-0.2μM滤膜过滤,内毒素<0.015EU/mL。然后,依次加入称量好的抗菌素、指示剂、碳酸氢钠,制得盐溶液;
2、检测步骤1所得盐溶液的渗透压和pH值,并记录最终渗透压和pH值;所述渗透压保持为260-285mOsm/Kg,所述pH值保持为7.30-7.80;
3、按照预配制的容量,将重组人血清白蛋白按终浓度为0.0-6.0mg/mL补充到盐溶液中;
4、将丙氨酸、天冬氨酸、天冬酰胺、谷氨酸、谷氨酰胺、甘氨酸、脯氨酸、牛磺酸、丝氨酸、苏氨酸、酪氨酸、甲硫氨酸、硫辛酸、精氨酸、胱氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、苯丙氨酸、苏氨酸、色氨酸、缬氨酸分别以0.01-1.00mmol/L的浓度溶解于盐溶液中;将透明质酸按照0.01-0.5mg/mL的浓度溶解于盐溶液中;将叶酸、肌醇、维生素B12、维生素B6、维生素B5、维生素B2、维生素B1、硫胺素、次黄嘌呤、泛酸分别以0.001-0.015mmol/L的浓度溶解于盐溶液中;将适量螯合剂溶解于盐溶液中;到充分溶解止;
5、将步骤4所得溶液经过0.2μm滤膜过滤灭菌后,取样测试;测试参数:
A、20-30℃条件下pH值:7.30-7.80;
B、渗透压:260-285mOsm/Kg;
C、内毒素:<0.15EU/mL;
D、一细胞鼠胚培养到96小时:≥80%的囊胚形成率;
6、将配制好的溶液在百级GMP车间进行无菌分装和标签。
【存储说明和稳定性】
1、存储:
把未开盖的培养液存放于2-8℃冰箱中。
使用前,在孵育箱温度(37℃)和在含5-6%CO2的大气环境中复温。不要把培养液冷冻或暴露于超过39℃的环境下。
把打开过的瓶放回冰箱前最好用含5-6%CO2的气体重新充气。特别强调要测定培养液在实验工作条件下的pH值,据此可调整使用CO2的浓度,以便获得胚胎发育需要最适合的pH值,一步培养液最佳pH值是7.30。
2、稳定性:产品在标签上有效期前是稳定的。
使用时,按照正确的无菌操作方法:
A.用无菌操作方法把适量的产品取出;
B.取出产品后不应把余下的倒回瓶中;
C.如产品变色,呈浊状,混浊或可能有微生物污染均不可使用。
【具体使用方法】
本发明所述的一步培养液主要用于受精后第1天至第5/6天胚胎胚的一步培养和胚胎移植。若一步培养液中已经加入重组人血清白蛋白(rHSA),可以随时使用。
胚胎的一步培养可以采用小滴、单孔皿或者四孔板内进行,并建议覆盖石蜡油,在拥有CO2培养箱的大气环境中进行体外培养。
用于胚胎移植时,可以将一步培养液置于单孔培养皿中。
Claims (6)
1.一种一步培养液,其特征在于:是将丙氨酸、天冬氨酸、天冬酰胺、谷氨酸、谷氨酰胺、甘氨酸、脯氨酸、牛磺酸、丝氨酸、苏氨酸、酪氨酸、甲硫氨酸、硫辛酸、透明质酸、精氨酸、胱氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、苯丙氨酸、苏氨酸、色氨酸、缬氨酸、叶酸、肌醇、维生素B12、维生素B6、维生素B5、维生素B2、维生素B1、硫胺素、次黄嘌呤、泛酸和螯合剂溶解于含有抗菌素和指示剂的盐溶液中配制而成;
所述一步培养液中,丙氨酸、天冬氨酸、天冬酰胺、谷氨酸、谷氨酰胺、甘氨酸、脯氨酸、牛磺酸、丝氨酸、苏氨酸、酪氨酸、甲硫氨酸、硫辛酸、精氨酸、胱氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、苯丙氨酸、苏氨酸、色氨酸、缬氨酸的浓度均为0.01-1.00mmol/L;透明质酸的浓度为0.01-0.50mg/mL;叶酸、肌醇、维生素B12、维生素B6、维生素B5、维生素B2、维生素B1、硫胺素、次黄嘌呤、泛酸的浓度均为0.001-0.015mmol/L;螯合剂适量。
2.根据权利要求1所述的一步培养液,其特征在于:所述的螯合剂为四元羧酸乙二胺四乙酸。
3.根据权利要求2所述的一步培养液,其特征在于:所述抗菌素为庆大霉素,浓度为4-12μg/mL。
4.根据权利要求3所述的一步培养液,其特征在于:所述指示剂为酚红,浓度4.5-10.0μg/mL。
5.根据权利要求4所述的一步培养液,其特征在于:所述盐溶液是以碳酸氢钠为缓冲剂,以钠、钾、镁、钙离子为基础,并以葡萄糖、丙酮酸钠、乳酸钠、白蛋白为能量物质的化合物溶液;其中包含:氯化钠89.00-108.00mmol/L、氯化钾4.00-6.00mmol/L、硫酸镁0.10-1.20mmol/L、磷酸二氢钾0.3-0.6mmol/L、磷酸二氢钠0.10-0.50mmol/L、氯化钙1.60-2.50mmol/L、碳酸氢钠18.00-26.00mmol/L、葡萄糖0.30-0.80mmol/L、丙酮酸钠0.20-0.60mmol/L、乳酸钠9.00-23.00mmol/L、磷酸二氢钾0.20-0.50mmol/L;
所述白蛋白为重组人血清白蛋白,是采用医疗级的重组人血清白蛋白干粉溶解于盐水溶液中制备而成,终浓度为0.0-6.0mg/mL。
6.如权利要求5所述一步培养液的制备方法,其特征在于:包括以下步骤:
(1)配制盐溶液:先按照盐溶液的各组分用量称量好各种组分,备用;再将除了抗菌素、指示剂、碳酸氢钠以外的各组分溶于超纯注射级用水中,溶解过程中遵循先固体后液体的原则;所述超纯注射级用水经0.1-0.2μM滤膜过滤,内毒素<0.015EU/mL;然后,依次加入称量好的抗菌素、指示剂、碳酸氢钠,制得盐溶液;
(2)检测步骤(1)所得盐溶液的渗透压和pH值,并记录最终渗透压和pH值;所述渗透压保持为260-285mOsm/Kg,所述pH值保持为7.30-7.80;
(3)按照预配制的容量,将重组人血清白蛋白按终浓度为0.0-6.0mg/mL补充到盐溶液中;
(4)将丙氨酸、天冬氨酸、天冬酰胺、谷氨酸、谷氨酰胺、甘氨酸、脯氨酸、牛磺酸、丝氨酸、苏氨酸、酪氨酸、甲硫氨酸、硫辛酸、精氨酸、胱氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、苯丙氨酸、苏氨酸、色氨酸、缬氨酸分别以0.01-1.00mmol/L的浓度溶解于盐溶液中;将透明质酸按照0.01-0.50mg/mL的浓度溶解于盐溶液中;将叶酸、肌醇、维生素B12、维生素B6、维生素B5、维生素B2、维生素B1、硫胺素、次黄嘌呤、泛酸分别以0.001-0.015mmol/L的浓度溶解于盐溶液中;将适量螯合剂溶解于盐溶液中;到充分溶解止;
(5)将步骤(4)所得溶液经过0.2μm滤膜过滤灭菌后,取样测试;测试参数:
A、20-30℃条件下pH值:7.30-7.80;
B、渗透压:260-285mOsm/Kg;
C、内毒素:<0.15EU/mL;
D、一细胞鼠胚培养到96小时:≥80%的囊胚形成率;
(6)将配制好的溶液在百级GMP车间进行无菌分装和标签。
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CN111019997A (zh) * | 2019-11-11 | 2020-04-17 | 浙江卫未生物医药科技有限公司 | 细胞活性检测模拟生理环境缓冲剂 |
CN115011552A (zh) * | 2022-07-01 | 2022-09-06 | 瑞柏生物(中国)股份有限公司 | 一种着床前胚胎发育用全程培养液及其制备方法 |
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CN115011552A (zh) * | 2022-07-01 | 2022-09-06 | 瑞柏生物(中国)股份有限公司 | 一种着床前胚胎发育用全程培养液及其制备方法 |
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