CN1625684A - 用于生物样品低温保藏的样品载具 - Google Patents

用于生物样品低温保藏的样品载具 Download PDF

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CN1625684A
CN1625684A CN03802972.3A CN03802972A CN1625684A CN 1625684 A CN1625684 A CN 1625684A CN 03802972 A CN03802972 A CN 03802972A CN 1625684 A CN1625684 A CN 1625684A
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乌尔里希·齐默尔曼
海科·齐默尔曼
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Abstract

本发明描述了一种用于低温保藏，特别是用于生物材料的低温保藏的方法和样品载具，其包括至少一个用于容纳生物样品的样品容器(2)，其中在样品容器(2)中设置由具有容积结构的材料制成的承载体(3)，该容积结构具有多个敞口的、可以填充样品的内部空腔。本发明也描述了一种机动车催化剂结构或生物形态陶瓷在制造用于在低温保藏时容纳样品的承载体中的用途。

Description

用于生物样品低温保藏的样品载具
本发明涉及一种用于生物样品低温保藏的样品载具(Probentrger)和方法。
在现代生物医药和生物技术中，对生物材料，特别是细胞、细胞群、天然或者人造组织、整个器官和胚胎机体的细胞进行低温保藏的需求在持续增长。在低温保藏下，人们将生物材料冷冻到低的、即所谓的低温温度(Cryogenic Temperature)，尤其是在-80℃～-196℃的温度范围。在此条件下，活细胞的新陈代谢实际上处于停滞状态，从而使细胞可以保存很多年。
为了保持低温保藏细胞的活性，人们使用了特定的低温介质(Cryomedia)，其包含防护材料(低温防护物)--例如二甲亚砜(DMSO)、乙二醇、甘油和糖，例如海藻糖或葡萄糖。低温防护物通过稳定细胞膜和大分子，以及通过防止细胞内冰的形成来保护细胞。细胞内冰晶对于细胞膜主要是产生不可逆的机械损害(见S.S.N.Murthy在“Cryobiology”第36卷，1998年，84-96页)。适合各种类型的细胞或组织的低温介质的最佳组合需实验确定。
除了低温介质的组合，对于保持比较高的细胞活性起着关键作用的是冷冻以及解冻模式(速度、温度梯度、持续时间等)。有关于此，迄今为止已经出版了关于不同样品种类以及保藏条件的大量规程，其既描述了快的冷冻以及解冻速度也描述了缓慢的冷冻以及解冻速度或者是两者的结合(参见，例如J.M.Baust等在“Cell Transplantation”第10卷，2001年，561-571页，以及M.Miyamoto等在“Cell Transplantation”第10卷，2001年，363-371页)。其中指出，在任一种情形下都需要精确控制的温度调节，因为细胞活性对冷冻/解冻速度依赖性很强。
如果将细胞悬浮液或者组织冷冻在传统的由塑料制得的低温容器中，不能保证样品中所有细胞都精确的遵循相同的冷冻速度。造成这种情况一方面由于不利的表面/容积比(低温容器/细胞样品)，另一方面由于低温容器普遍采用的塑料材料和水性的低温介质具有很小的导热能力。
这不仅导致冷冻的样品内部陡直的温度梯度，而且造成冷冻速率强烈的空间不均匀性。根据它所处位置的不同温度条件，每一个细胞得到不同冷冻或解冻速率。因此，在低温速率下强烈的不均匀性对于单个细胞而言造成活性的严重丧失，因为只有所有细胞遵循最佳冷冻速率，样品才具有高的活性。
为了避免或者消除这些问题，已知以下解决途径。由Berison等(参见“Anim.Reprod”第63卷，2000年，205-220页)证实：在冷冻猪的精液时，相对于在圆柱状塑料管(所谓的Maxi-straws)中冷冻，在平的塑料容器(所谓的扁平包装)中冷冻，精液活性可以得到显著提高。作者认为其原因可归结为平的低温容器中的均匀的冷冻和解冻过程。另一个问题是，在“Maxi-straws”中，由于在样品周围已经解冻的水的隔离特性，导致样品内核中解冻速度严重减慢。
由S.-P.Park等(参见“Human Reproduction”第15卷，2000年，1787-1790页)描述了将人体胚胎超快冷冻在最初为电子显微镜而设计的铜格栅中。在这种情况下，当将样品浸入液氮中时，热量可被非常迅速地带走，而这对于胚胎活性而言是有利的。
由实践中，人们知道用于保存微生物的商品化的系统(产品“Micrybank”，制造者：SenseLab-Gmbh，莱比锡，德国)。其将微生物结合在小陶瓷球的多孔表面上，在特定的低温介质中保藏。然而，这个系统仅是对样品的改进处理。冷冻过程不理想。陶瓷球具有比较小的热传导性，从而能够阻止快的冷冻或解冻。此外，细胞或者组织由于它们的尺寸而不适合于吸附到多孔表面上。
少量样品体积(毫升或微升范围)形式的生物样品冷冻在适当结构的二维冷冻基片上也是已知的。由此可以避免所述的温度梯度以及冷冻速度梯度的问题。借助这项技术，仅能使少量样品放置在冷冻基片上，这在需要大体积样品(例如在毫升范围)的应用中是个缺陷。
本发明的目的在于可以提供用于生物样品低温保藏的改进的样品载具，借此可以克服传统样品载具的缺点。根据本发明的样品载具应该使得、尤其是能够可再生地调节所定义的低温或解冻规程，其中应该能够避免样品内温度梯度或者冷冻速度或者解冻速度的变化。此外，该样品载具应该适合于大体积样品的保藏。本发明的目的还在于可以提供一种改进的低温保藏方法，借此可以避免传统的冷冻或解冻规程的缺点。
该目的通过具有根据权利要求1和7的特征的样品载具和方法实现。本发明的优选的实施方案和应用由其从属权利要求给出。
根据本发明的第一个重要方面，提供了一种用于低温保藏、特别是用于生物样品低温保藏的样品载具，其具有至少一个样品容器以容纳生物样品，其中设置由具有以下结构的材料得到的承载体(Trgerkrper)，该结构具有多个内部的、可填充样品的敞口空腔。带有内部空腔的承载体具有比样品容器的内表面大得多的内表面。由此得到的表面/容积的比例相对于传统的样品载具有利地得到显著改善。通过借助承载体与低温介质的热传导连接，冷冻和解冻过程在温度分配和温度变化的速度方面能够实现显著均匀化。
根据本发明的优选实施方案，所述承载体至少部分地由具有像金属一样的导热性材料组成。据此，冷却剂实际的温度能够以更快的速度传输到样品上。有利地，能够以更高的准确性和可再现性进行冷冻和解冻规程。承载体优选地由金属、金属合金或者包括金属或金属合金的复合材料组成。承载体也可由聚合物材料或者陶瓷材料构成，其内表面镀有具有与金属导热率相应的导热能力的金属、金属合金、半导体材料或者化学粘合剂。聚合物可以例如通过合成塑料或者基于天然有机材料的、特别是基于纤维素的固定材料，其例如通过热解的木材而制得。
带有内空腔的结构优选地由其空间遍布蜂窝、孔道、沟槽或者其它可穿通结构的结构体构成。空腔的最小横截面尺寸有利地在1μm-10mm范围中，优选地在5μm-5mm范围中。空腔可以设置成有规则(蜂窝结构)或者无规则结构(内部镀膜的植物或者动物组织或者器官、海绵或者泡末结构)。规则布置具有以下优点：在承载体中吸收的样品体积可以定量地准确得出。不规则结构通常可以根据承载体的制造方法不同，以良好的表面/容积比生产。
具有内表面的承载体在其外形上优选地与本发明的样品载具的至少一个样品容器的内部形状相适应。这个特点的优点在于具有样品容器空间高利用率以及整个样品容器中的低温保藏条件具有均匀性。承载体可以固定的或松动的方式设置在样品容器中。固定安装具有以下优点，即本发明的样品载具具有紧凑的、独立的结构，特别是在自动系统中可以毫无问题地使用。如果承载体可从样品容器中抽出，这将在承载体的清洗和/或预处理(例如预冷冻)上带来优点。
本发明的另一个优点在于，承载体可以用作热交换器，例如当样品容器仅部分填充，使得仅承载体的一部分空腔结构装有样品，这样剩下的上方的结构可用作热交换器。例如蒸汽状的低温介质掠过承载体的空闲部分并且以此加快了热量的排出，反过来，在解冻过程中也可加快热量的供给。根据优选的实施方案，承载体可以附加配备远离承载体并伸进低温介质中去的热交换元件。通过这种布置，热交换在完全填满的承载体中也得到改善。
根据本发明的一个优选的实施方案，承载体由传统的催化剂、比如在机动车中使用以减少有害物质的催化剂的载体材料制得。包含催化剂结构的承载体由例如以贵金属(例如铂)镀膜的钢或陶瓷的基本材料组成。采用催化剂结构具有特别的优点，即已知的催化剂载体在大的内表面方面已经得到优化并且由适合于在低温保藏中使用的惰性材料组成。
根据本发明的又一优选实施方案，承载体由所谓的由生物形态陶瓷组成，所述生物形态陶瓷由基于多糖的材料制得。优选地，这种材料可以由植物制得，根据植物的生长特性不同，可以得到具有任一期望尺寸的空腔。生物形态陶瓷的又一个优点在于其生物相容性。
本发明的主题还在于一种应用所述载具进行低温保藏的方法。通过本发明方法可利用的技术——例如吸移或者倾析(decanting)，可以将生物样品加到承载体上，并被承载体容纳。根据本发明方法的优选实施方案，采用一个预冷冻的承载体，样品由此优选地在真正的冷冻过程之前经历均匀的预冷冻。
根据本发明保藏的生物样品一般包括含有生物材料的液体。该液体包括例如培养基、营养溶液或者其他兼容的用来包覆的介质。本发明优选的可以使用感兴趣的生物材料，例如细胞、细胞群、天然或人造组织、组织切片、器官、部分器官或者胚胎机体的细胞。样品可以涉及例如加入了生物材料的悬浮样品或液体。因此，样品也可以包括其中生物材料例如处于容器底部的液体。材料的尺寸一般和载具中空腔的尺寸彼此相一致。这使得用于提供给组织生产(“组织工程”)的生物组织或者组织的部分能够有利地得到保藏。
本发明的其它优点和特点在以下参照附图描述，图中所示为：
图1和2：根据本发明样品载具的若干实施方案的示意图；和
图3和4：以根据本发明的样品载具得到的实验结果。
根据本发明的样品载具1包括对应于图1所示实施方案中的样品容器2，其中设置承载体3。样品载具1具有例如底部封闭的圆柱的形状。其例如可以由已知的小样品管或者试管构成。承载体3具有较大内表面的容积结构。承载体3的外形与样品容器内部形状相适应或者比其稍大(参见下面)。由承载体3占据的样品容器2的内部空间处于例如0.5-10ml的范围，优选地为1-5ml。
承载体3由具有高导热率的多孔的、类似蜂窝的、海绵状或者泡沫状的金属材料，或者由内部镀膜的植物或动物组织或器官构成。优选地，承载体能够实现准确、快速、可调的热量导进、排出。导热率总计为例如至少0.1J/K kg。内表面与承载体的容积比取决于所用承载体材料的种类和/或其生产条件，在具体应用中，承载体材料或者所说的表面/容积比根据要保藏的样品的性质(例如悬浮样品)以及供利用的低温设备来选择。该比例可以总计为例如4000m-1，承载体的内部空腔由其厚度优选地小于0.2-0.5mm的壁来分隔。
承载体3例如由用于机动车的金属催化剂材料形成。孔道大小或者单位体积的细胞数根据要保藏的不同的生物样品来选择。承载体3例如由铂(导热率为0.32J/K kg)、钢(0.11J/K kg)或者铝(0.22J/K kg)组成。承载体3例如由用于机动车的、由德国Fuerstenfeldbruck制造商FA.Matrix制造的金属催化剂或者陶瓷-金属-催化剂，剪切得到。
传统催化剂结构的优点在于其较小的热膨胀，使得在保藏过程的不同阶段基本保持承载体的外部尺寸。
或者，承载体可以由聚合物或者陶瓷构成的复合材料组成，其具有由良好热传导材料构成的内部镀膜。此外，承载体可以自基于天然多糖的材料(例如纤维素、藻酸盐、果胶)制备，特别是基于木材或者植物纤维制备，其中该材料经过热解过程和内表面的处理，如硅烷化或者金属沉积(所谓的生物形态陶瓷)。具有多孔结构的硅烷化木材特别适合作为承载体，如P.Greil在“Journal of the European Ceramic Society”第21卷，2001年，105-188页中描述的。其他用于生物相容材料的植物或者动物原料的范例包括基于仙人掌、大戟属植物、硅藻、纸莎草芦苇、海藻、蜀葵、接骨木、压制动物皮、骨头尤其是管状骨头、海绵和蜂窝的承载体。或者，承载体可以由天然的多孔矿石，例如鼓气陶土，通过内部镀膜制造。
由生物形态陶瓷制得的承载体按照以下方法制造。首先，将木材本体热解以形成相应的由碳构成的引导束支架(Guiding BundleFramework)。例如在1400℃时进行聚合。接着，引导束支架浸泡在镀膜溶液中。镀膜溶液的液体浸湿引导束支架的内表面。镀膜例如在硅熔液中在约1600℃时进行。在从熔液中抽出的承载体的冷冻过程中，在其内表面沉积硅层。有利的，根据所选用的木材，可以制得不同孔道尺寸的承载体。此外，承载体的外部尺寸有大的可变性。硅烷化的竹子提供例如具有直径约1-5cm的承载体。利用藤条可以制得直径达大约1cm的承载体。
可以用于代替硅烷化的是，可以用具有生物相容的、无毒金属对热解引导束支架(Xylem)进行镀膜。可以使用例如金熔液或者铂熔液作为镀膜溶液。
承载体的内表面一般经过多次镀膜。例如，在生物形态陶瓷中，由硅、金以及其它生物相容材料构成一种多层镀膜。一般采用如已知的晶体镀膜半导体技术的技术以在承载体内表面镀膜。
承载体的另一个优点在于它的机械可变形性。虽然承载体由固定材料制造，但由于具有强的可透气的容积结构，它的外形可轻易地弹性变形。首先，可以制备其外形比样品容器稍大的承载体。在将承载体装入样品中时，承载体受到挤压，使得在弹性变形作用下固定在样品容器中。
图2表示具有细胞培养板形状的、包含多个样品容器21、22、23…的样品载具1，其中分别放置有一个承载体31、32、33…。这些样品载具具有例如圆柱形状。图2的上部表示承载体的俯视图。不同模式的部件图A、B和C展示根据本发明使用的承载体可以配备的空腔的不同形状。
例如，根据图2A的承载体由波浪形金属片组成，其移动或者在使用时其间排列的平面彼此连接，从而形成多个直通道41，通道41沿承载体的轴向通过。根据图2B的承载体具有无序的蜂窝结构。图2C是有序的蜂窝结构，同样具有轴向延伸的通道43。
图2A示意地表示出悬浮样品10的填充高度。承载体31高于该高度的闲置区域需要与周围进行热交换。为了进一步改进在承载体全部填充时的热交换，可以至少配备一个突出的热交换元件51。热交换元件51例如由与承载体相同的材料组成。例如，可提供一个突出的设置成突入空间中的或者在样品容器壁的表面上的条状物。
如图2B、2C所示，可以采用热交换元件52、53作为承载体样品容器之间的连接。通过热交换元件52、53，可以由多个承载体32、33构成复合结构，借此可以同时配备大量样品容器。根据本发明，例如对应于微滴定板或纳滴定板的尺寸设置并且通过热交换元件彼此相连的承载体形成承载体复合结构。
将冷却剂与根据图1或图2的样品载具1接触以进行低温保藏，其借助承载体直接进行热交换或者经过样品容器的壁材料和/或热交换元件的帮助间接联接。
实施例
图3和图4中所示的试验结果在以下实验条件下确定。对呈指数增长状态的细胞系SP2的细胞进行低温保藏。使用90％FCS和10％DMSO组成的混合物或者含海藻糖的溶液作为低温介质。使用根据图2的具有6个样品容器的细胞培养板作为样品载具。在每一容器中使用由催化剂材料得到的、具有约0.5*2*2cm3体积的圆片。该承载体以酒精消毒并且在它的底边用塑料膜封住。
为了制备悬浮样品，首先，将SP2细胞收集到培养瓶中并且借助CASY测量分析细胞大小及细胞数量。形成8.2*105细胞/ml的悬浮样品。悬浮样品在低温保藏之前约10分钟以1000rpm进行离心分离。这种情况下形成的细胞球团在每1ml预冷的冷却剂中重新悬浮，并吸移进承载体中。
可以实现不同的冷冻和解冻规程。快速冷冻意味着即时转换到-80℃的箱中。缓慢冷冻意味着在-20℃的箱子中预冷冻1小时，接着转换到-80℃的箱中。快速解冻意味着直接从箱子的-80℃转换到环境温度。缓慢解冻包括首先在-20℃的箱子中持续1小时的升温，接着在10ml CGM(37℃)的吸移量下在冰上保存30分钟，而后反复进行样品的重新悬浮。
为了检验低温保藏结果，对解冻的样品进行离心分离，并且在CGM中重新悬浮，在37℃和5％的CO2条件下培养24小时后，进行CASY测量以计算低温保藏之后的细胞数量、细胞大小以及细胞活性。图3和图4表示对于不同冷冻和解冻规程，将根据本发明的样品载具与对照样品(在试管中低温保藏)相比较的结果。对于所有实验条件，尤其是快速冷冻过程，根据本发明的样品载具的低温保藏可以显著地提高细胞的活性以及活细胞的数量。

Claims (13)

1、一种用于低温保藏、特别是用于生物材料低温保藏的样品载具，包括至少一个用于容纳生物样品的样品容器(2，21，22，23)，其特征在于，在所述样品容器(2，21，22，23)中设置由具有容积结构的材料制成的承载体(3，31，32，33)，该容积结构具有大量敞口的、可以填充样品的内部空腔(41，43)。
2、根据权利要求1的样品载具，其中所述承载体(3，31，32，33)由导热率与金属导热率相同的材料组成。
3、根据权利要求1的样品载具，其中所述承载体由复合材料组成，其中内部空腔至少部分镀有导热率与金属导热率相同的材料。
4、根据前述权利要求中任一项的样品载具，其中所述承载体(3，31，32，33)具有蜂窝、孔道、沟槽或者类似海绵或泡沫状的空腔。
5、根据前述权利要求中任一项的样品载具，其中所述承载体(3，31，32，33)的形状与所述样品容器(2，21，22，23)的内部形状相适应或者稍大。
6、根据权利要求5的样品载具，其中所述承载体(3，31，32，33)在弹性变形作用下紧密安装在所述样品容器(2，21，22，23)中。
7、根据前述权利要求中任一项的样品载具，其中所述承载体(3，31，32，33)由机动车的催化剂材料制成。
8、根据权利要求1-5中任一项的样品载具，其中所述承载体(3，31，32，33)由生物形态陶瓷制成。
9、根据前述权利要求中任一项的样品载具，其配备至少一种热交换元件(51，52，53)。
10、根据权利要求9的样品载具，其具有多个样品容器，并且各个所属的承载体通过热交换元件(52，53)彼此连接。
11、一种低温保藏方法，尤其是生物材料的低温保藏方法，其中将生物样品加入到根据前述权利要求中任一项的样品载具中，并且使所述样品载具经历冷冻过程。
12、根据权利要求11的方法，其中使用预冷的承载体。
13、一种机动车催化剂结构或生物形态陶瓷在制造用于在低温保藏时容纳样品的承载体中的用途。
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