CN117427219A - 脱细胞带瓣管道制备方法 - Google Patents
脱细胞带瓣管道制备方法 Download PDFInfo
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Abstract
本发明提供了一种同种带瓣管道及其保存方法,属于医疗技术领域;所述同种带瓣管道取自于心脏移植的受体置换下来的新鲜心脏。传统来源的同种带瓣管道常取自遗体捐献,本发明的同种带瓣管道取自于新鲜的人源性心脏供体,拓展了带瓣管道材料选择的范围,提高了带瓣管道的血管顺应性和同天然带瓣管道的相似性,减少了处理过程中抗生素消毒对带瓣管道的损伤。本发明的同种带瓣管道具有使用前无需抗生素处理,且冷缺血时间短的优点。本发明还提供了上述方案所述同种带瓣管道的保存方法,本发明的保存方法改进了带瓣管道的保存流程,选择快速冷冻,最大化降低保存过程中的细胞损伤。本发明的保存方法能够提高带瓣管道的使用寿命。
Description
技术领域
本发明属于医疗技术领域,具体涉及一种同种带瓣管道及其保存方法。
背景技术
法洛四联症是联合的先天性心血管畸形,主要有四种心脏结构的畸形:室间隔缺损、肺动脉狭窄、主动脉骑跨、右心室肥厚。法洛四联症是常见的先天性心脏病,在紫绀型先天性心脏病中居首位。手术是法洛四联症首选的治疗方式,对于合并肺动脉闭锁的严重畸形患者,需要使用带瓣管道重建右心室流出道以纠正异常的血流动力学,带瓣管道是影响手术效果的最重要因素。
带瓣管道分为同种带瓣管道(valved homograft conduit)、异种带瓣管道(valved heterograft conduit)和人工合成带瓣管道。其中,异种带瓣管道来自异种动物,虽然不受供体选择范围的限制,但免疫排斥程度高。其血栓形成风险和术后感染风险高于同种带瓣管道,且中长期临床结局和再次手术率均差于同种带瓣管道。人工合成带瓣管道创建复杂3D结构的能力有限,且目前可用的组织工程带瓣导管均不具有与天然组织相同的材料特性,尤其是各向异性特性,因此人工合成材料带瓣管道的生物相容性和血流动力学性能均不如同种带瓣管道。同种带瓣管道符合生理特点,具有良好的血流动力学特性,生物相容性好,不易钙化,对感染抵抗力高且无需终生抗凝,在临床上有着重要应用价值。
目前同种带瓣管道主要来源于遗体捐献。但是,遗体捐献来源的带瓣管道往往不能保证无菌,使用前需要进行消毒处理。但是,消毒处理常用的抗生素如两性霉素B具有细胞毒性,因此消毒处理有可能导致同种移植瓣膜生物活性丧失,瓣膜易发生变性、术后钙化、远期效果差。此外,对于长时间死亡的患者,热缺血时间>24h时,同种带瓣管道内皮细胞基本死亡脱落,前列环素的合成停止,动脉壁中的胶原纤维被破坏,结构完整性也被破坏,与新鲜血管相比,血管组织力学在超过24h后显着降低。因而,目前同种带瓣管道的广泛应用受到限制。
发明内容
本发明的目的在于提供一种同种带瓣管道及其保存方法,本发明的同种带瓣管道具有使用前无需抗生素处理,且冷缺血时间极短的优点。
本发明提供了一种同种带瓣管道,所述同种带瓣管道取自于心脏移植的受体置换下来的新鲜心脏。
优选的,所述新鲜心脏的冷缺血时间≤24h。
本发明还提供了上述方案所述同种带瓣管道的保存方法,包括以下步骤:
将所述同种带瓣管道置于装有冻存保护液的冻存袋内,于-70至-90℃的条件下冻存12~24h后,转入液氮保存。
优选的,所述冻存保护液以M199培养基为基础,还包括体积浓度为10%~15%的二甲基亚砜。
优选的,所述冻存袋的层数为多层。
优选的,在进行所述冻存前,还包括对同种带瓣管道取样检测是否存在细菌污染;在所述检测的过程中,盛装有所述同种带瓣管道的冻存袋置于4℃条件下保存;所述4℃条件下保存的时间≤60min。
优选的,在转入液氮保存后,还包括解冻;所述解冻的温度为37℃;所述解冻的时间为10~30min。
优选的,所述解冻包括在生理盐水中水浴解冻。
优选的,在所述解冻后,还包括将解冻后的同种带瓣管道浸泡于M199培养基中。
优选的,所述浸泡的次数为3次,每次浸泡的时间为5min。
本发明提供了一种同种带瓣管道,所述同种带瓣管道取自于心脏移植的受体置换下来的新鲜心脏。和商业公司常用的尸源性带瓣管道不同,本发明的同种带瓣管道取自于新鲜的人源性心脏供体,拓展了带瓣管道材料选择的范围,扩大了带瓣管道移植选择库,提高了带瓣管道的血管顺应性和同天然带瓣管道的相似性,减少了处理过程中抗生素消毒对带瓣管道的损伤。本发明的同种带瓣管道具有使用前无需抗生素处理,且冷缺血时间极短的优点。
本发明还提供了上述方案所述同种带瓣管道的保存方法,本发明的保存方法改进了带瓣管道的保存流程,选择快速冷冻,最大化降低保存过程中的细胞损伤。本发明的保存方法能够提高带瓣管道的使用寿命和临床效果。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动性的前提下,还可以根据这些附图获得其他的附图。
图1为实施例1的流程图。
具体实施方式
本发明提供了一种同种带瓣管道,所述同种带瓣管道取自于心脏移植的受体置换下来的新鲜心脏。
本发明的同种带瓣管道应用了新鲜的人源性心脏供体,拓展了带瓣管道材料选择的范围,提高了带瓣管道的血管顺应性和同天然带瓣管道的相似性,减少了处理过程中抗生素消毒对带瓣管道的损伤。并且,本发明的同种带瓣管道可以根据受体心脏进行个性化定制,修剪适合长度的带瓣管道,清洗过程中避免直接使用抗生素对瓣膜损伤。
在本发明中,对所述心脏移植的受体存在1)~4)所述要求:
1)无结核、肝炎、梅毒、艾滋、疟疾、麻风等传染病,败血症和肿瘤病史;排除大三阳、小三阳、单HBsAg阳性;单HBsAb阳性不排除、单HBcAb阳性或HBcAb+HBsAb阳性不排除,但需要备注;
2)无大面积烧伤、自身免疫病、心脏外科手术史、瓣膜病、心肌病、未经治疗的活动性肺疾病、风湿热、细菌性心内膜炎、经历过长时间闭式CPR和病因不明的严重疾病;
3)年龄不足60岁,50岁以上需要备注。
在本发明中,所述新鲜心脏的冷缺血时间≤24h。
本发明还提供了上述方案所述同种带瓣管道的保存方法,包括以下步骤:
将所述同种带瓣管道置于装有冻存保护液的冻存袋内,于--90~-70℃的条件下冻存12~24h后,转入液氮保存。
相对常规以每分钟降温1℃的限速冷却的冻存条件,本发明对同种带瓣管道于-80℃的条件下直接冻存12~24h,在冻存过程中,每分钟平均降温4~5℃,冻存时间缩短,不利于冰晶形成,最大化降低保存过程中的细胞损伤。并且,本发明的保存方法无需进行预冷却,也能够降低细胞损伤。
在进行冻存前,本发明优选的还包括同种带瓣管道取样检测是否存在细菌污染,如出现细菌污染则该带瓣管道不可用;在所述检测过程中,盛装有所述同种带瓣管道的冻存袋优选的于4℃条件下保存;所述于4℃条件下保存的时间优选的≤60min,更优选为30~60min。
在本发明中,所述冻存保护液优选的以M199培养基为基础,还包括体积浓度为10%~15%的二甲基亚砜(DMSO);更优选的,所述冻存保护液由M199培养基和DMSO组成,所述冻存保护液中DMSO的体积浓度为10%。本发明应用M199培养基+DMSO二甲基亚砜的冻存保护液代替传统含丙二醇的冻存保护液,减少了生物毒性。
在本发明中,所述冻存袋的层数优选为多层,更优选为3层;所述冻存袋优选的包括无菌塑料袋。在本发明中,所述同种带瓣管道优选的封装于装有冻存保护液的冻存袋的最内层。
将所述同种带瓣管道置于装有冻存保护液的冻存袋内后,本发明优选的采用冰盒转运同种带瓣管道进入-90~-70℃条件下冻存,更优选的于-80℃条件下冻存。
本发明于-90~-70℃条件下进行快速冷冻,使温度快速经过相变点,令细胞内液体成分快速转变为玻璃态,降低冰晶形成。
在转入液氮保存后,本发明优选的还包括还包括解冻;所述解冻的温度为37℃;所述解冻的时间为10~30min,优选为15~20min。
在所述解冻前,且上述方案所述冻存袋为3层时,本发明优选的还包括打开外侧两层包装袋后浸泡碘伏,之后再进行解冻。
相比常规的于常温(20~30℃)条件下解冻,本发明设置解冻温度为37℃,能够缩短解冻时间,达到快速复温的效果,减少冰晶形成,进而减少细胞损伤。
在本发明中,所述解冻优选的包括在生理盐水中水浴解冻。
在所述解冻后,本发明优选的还包括将解冻后的同种带瓣管道浸泡于M199培养基中。
在本发明中,所述浸泡的次数优选为3次,每次浸泡的时间优选为5min。
在所述浸泡后,本发明优选的还包括将浸泡后的同种带瓣管道放入新的M199培养基中交给器械护士等待移植。
为了进一步说明本发明,下面结合附图和实施例对本发明提供的一种同种带瓣管道及其保存方法进行详细地描述,但不能将它们理解为对本发明保护范围的限定。
实施例1
流程图参见图1。
1.设备和耗材:
1.1.设备:层流生物安全柜,液氮保存罐,4℃冰箱,-80℃低温冰箱,热合机
1.2.耗材和试剂:细菌培养瓶,耐低温聚乙烯冻存袋,0.9%生理盐水500ml/袋,50mL注射器,M199培养基,DMSO
2.准备工作:
2.1.选择供体:供体来自于参与心脏移植手术的受体新鲜心脏,患者标准遵循下列要求:
(1)无结核、肝炎、梅毒、艾滋、疟疾、麻风等传染病,败血症和肿瘤病史(排除大三阳、小三阳、单HBsAg阳性,单HBsAb阳性不排除,单HBcAb阳性或HBcAb+HBsAb阳性不排除,但需要备注);
(2)无大面积烧伤、自身免疫病、心脏外科手术史、瓣膜病、心肌病、未经治疗的活动性肺疾病、风湿热、细菌性心内膜炎、经历过长时间闭式CPR和病因不明的严重疾病;
(3)排除核酸阳性;
(4)年龄不足60岁,50岁以上需要备注;
(5)冷缺血时间≤24h。
2.2.消毒:
(1)提前准备各个耗材,送至环氧乙烷室消毒;
(2)提前准备取材器械和对应包装包经供应室高温高压灭菌;
(3)操作前50min,层流生物安全柜及操作间开紫外线照射20min,关紫外线、开风机吹10min。
2.3.取带瓣管道前准备:
(1)术者取下心脏,装入无菌塑料袋内(单层),由助手从手术间移至细胞间内;
(2)修剪者刷手入细胞间,在助手协助下穿手术衣,带无菌手套;
(3)打开铺料包,取中单及治疗巾由外向内、由左向右依次铺放4~6层;
(4)助手打开器械包外层包装,由修剪者打开内层包装,将不锈钢桶与器械取出,置于层流生物安全柜无菌台上,摆放整齐。打开2袋500mL 0.9%生理盐水倒入不锈钢桶内备用;
(5)助手将层流生物安全柜操作窗拉下、留30cm高;
(6)助手双手提装有供心的包装袋边缘,将包装袋口外翻(注意不可触碰包装袋内侧);修剪者将供心取出,放入灭菌后的弯盘内;
(7)2ml注射器抽取内层塑料袋中剩余液体,盖好针头及防护帽放至一旁;
3.带瓣管道制备:
3.1.心脏清洗和修剪带瓣管道:
(1)0.9%生理盐水冲洗心脏,轻轻挤压排空心腔内血液及盐水,一手轻扶心脏、一手端起冲洗盆将冲洗液倒入医疗垃圾袋;重复2次;
(2)分离二尖瓣、三尖瓣乳头肌,沿瓣环下5~10mm剪开心室肌、瓣环上5~10mm剪开心房、主肺动脉瓣环下5~10mm剪开心室肌,将剪下的心室肌和心房交给助手;
(3)从不锈钢筒中向弯盘中倒50ml生理盐水,剔除主动脉外膜、小心分离左右冠脉分支,保留5mm剪断左右冠脉;
(4)游离主肺动脉交界处,将主肺动脉带瓣管道分开,肺动脉带瓣管道放入不锈钢筒浸泡,不锈钢筒随时加盖。
(5)取出肺动脉带瓣管道,小心剔除外膜,修剪瓣环下心肌,保留5mm,剔除心肌外脂肪,放入不锈钢筒浸泡;
(6)取不锈钢尺,测量主肺动脉长度、瓣环上半周长,二尖瓣前后、左右径、前瓣直径、乳头肌腱索长度,三尖瓣瓣叶直径、乳头肌腱索长度,由助手逐一记录,尤其是有无动脉壁破损和冠脉钙化。
3.2.冻存带瓣管道:
(1)修剪者更换无菌手套,在层流生物安全柜上加铺2层铺料,取出冻存袋和2支50mL注射器备用;
(2)取M199培养基,酒精消毒瓶口,酒精灯外焰过火2~3圈;
(3)取DMSO,酒精消毒瓶口,酒精灯外焰过火2~3圈;
(4)向生物安全柜内侧打开M199培养基,注射器抽取45mL;向生物安全柜内侧打开DMSO,注射器抽取5mL,加入烧杯,配置体积浓度为10%的DMSO冻存保护液;
(5)用无损伤镊依次取出瓣膜,剪小块动脉壁放入不锈钢桶内;
(6)将瓣膜分别放入最内层无菌塑料袋里,加20~30ml冻存保护液,排出袋内空气,向内折袋口塑封条2~3圈,将无菌塑料袋密封,放入第2层无菌塑料袋内,在袋口处罩隔热膜,放至热合机处,连续启动3次;
(7)修剪者带隔热膜取出包装,5s后捏住朝向塑料袋的一面撕掉隔热膜,拿回生物安全柜观察有无漏液;
(8)剪掉封口处多余塑料袋,装入第3层无菌塑料袋,热合机封口,冷却后取出;
(9)低温记号笔在最外层包装袋上标记瓣膜编号及日期,同时贴橡皮膏标记,放入4℃冰箱暂存30~60min;
(10)将最初抽取的取材液与留取小块动脉壁分别注入细菌培养瓶关紧,送至培养室,如出现细菌污染则该带瓣管道不可用;
(11)冰盒转运带瓣管道进入-80℃冰箱保存;
(12)12h后,瓣膜由深低温冰箱转入液氮保存,在液氮罐上标记瓣膜编号,日期,记录时间,液氮罐位置。
3.3.取用带瓣管道:
(1)在手术室无菌台上取出冻存袋,打开外侧两层包装袋,先浸泡碘伏后,放入37℃盐水中解冻;
(2)初步解冻完成后,M199培养基浸泡5分钟,重复三次;
(3)检查瓣膜有无问题,放入新的M199培养基中交给器械护士等待移植。
对比例1
采用异种生物瓣膜美敦力Freestyle无支架生物瓣膜(Medtronic Inc.,Minneapolis,MN,USA)进行移植。
实施例1和对比例1的带瓣管道移植效果对比数据参见表1。从表1可以看出,相对于实施例1的新鲜同种带瓣管道,对比例1的异种生物瓣膜术后更容易发生瓣膜衰败和随访中死亡。
表1接受新鲜同种带瓣管道和异种生物瓣膜术后临床结局
对比例2
采用尸检来源同种带瓣管道进行移植。
实施例1和对比例2的带瓣管道移植效果对比数据参见表2。由表2可以看出,实施例1的移植来源同种带瓣管道和对比例2的传统来源同种带瓣管道均对右室功能具有改善效果,移植来源同种带瓣管道的效果不劣于传统来源同种带瓣管道,扩大了器官移植供体来源,此外在RVEDD的改善程度上优于传统来源。
表2实施例1中新鲜移植同种带瓣管道和传统来源带瓣管道移植后效果对比
对比例3传统控温冷冻同种带瓣管道
传统控温冷冻的程序为:-135℃甲基丁烷浴15min后,-1℃/min冷却到-80℃。
实施例1和对比例3的带瓣管道移植效果对比数据参见表3。由表3可以看出,快速冷冻相对于控温冷冻方法,更少出现心血管不良事件、瓣膜狭窄或反流。
表3快速冷冻(实施例1)和传统控温冷冻带瓣管道移植后临床结局对比
对比例4传统复温方式带瓣管道
传统复温方式带瓣管道采用常温(20~30℃)解冻,其余同实施例1。
实施例1和对比例4的带瓣管道移植效果对比数据参见表4。由表4可以看出,37℃快速解冻,相对于常温解冻瓣膜更不容易发生狭窄和反流。
表4快速复温(实施例1)和传统复温方式带瓣管道移植后临床结局对比
尽管上述实施例对本发明做出了详尽的描述,但它仅仅是本发明一部分实施例,而不是全部实施例,人们还可以根据本实施例在不经创造性前提下获得其他实施例,这些实施例都属于本发明保护范围。
Claims (10)
1.一种同种带瓣管道,其特征在于,所述同种带瓣管道取自于心脏移植的受体置换下来的新鲜心脏。
2.根据权利要求1所述的同种带瓣管道,其特征在于,所述新鲜心脏的冷缺血时间≤24h。
3.权利要求1或2所述同种带瓣管道的保存方法,其特征在于,包括以下步骤:
将所述同种带瓣管道置于装有冻存保护液的冻存袋内,于-70至-90℃的条件下冻存12~24h后,转入液氮保存。
4.根据权利要求3所述的保存方法,其特征在于,所述冻存保护液以M199培养基为基础,还包括体积浓度为10%~15%的二甲基亚砜。
5.根据权利要求3所述的保存方法,其特征在于,所述冻存袋的层数为多层。
6.根据权利要求3所述的保存方法,其特征在于,在进行所述冻存前,还包括对同种带瓣管道取样检测是否存在细菌污染;在所述检测的过程中,盛装有所述同种带瓣管道的冻存袋置于4℃条件下保存;所述4℃条件下保存的时间≤60min。
7.根据权利要求3所述的保存方法,其特征在于,在转入液氮保存后,还包括解冻;所述解冻的温度为37℃;所述解冻的时间为10~30min。
8.根据权利要求7所述的保存方法,其特征在于,所述解冻包括在生理盐水中水浴解冻。
9.根据权利要求7所述的保存方法,其特征在于,在所述解冻后,还包括将解冻后的同种带瓣管道浸泡于M199培养基中。
10.根据权利要求9所述的保存方法,其特征在于,所述浸泡的次数为3次,每次浸泡的时间为5min。
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