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Jan 2018
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Maintenance of Schmidtea mediterranea in the Laboratory
实验室真涡虫的保存   

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Abstract

In the last years, planarians have emerged as a unique model animal for studying regeneration and stem cells biology. Although their remarkable regenerative abilities are known for a long time, only recently the molecular tools to understand the biology of planarian stem cells and the fundamentals of their regenerative process have been established. This boost is due to the availability of a sequenced genome and the development of new technologies, such as interference RNA and next-generation sequencing, which facilitate studies of planarian regeneration at the molecular and genetic level. For these reasons, maintain a healthy and stable planarian population in the laboratory is essential to perform reproducible experiments. Here we detail the protocol used in our laboratory to maintain the planarian species Schmidtea mediterranea, the most widespread as a model.

Keywords: Planarian (真涡虫), Schmidtea mediterranea (真涡虫), Culture (培养), Planarium (真涡虫), Model organism (模式生物), Regeneration (再生)

Background

Planarians are bilaterally symmetric platyhelminthes, members of the superphylum lophotrochozoa. There are terrestrial, marine, and freshwater planarians. They prey predominantly upon injured insects, insect larvae, and other invertebrates. Planarians are triploblastic and acoelomated animals that lack circulatory, skeletal, and respiratory systems (Figure 1A). These animals have the amazing ability to restore any missing part of their body after an amputation in a few days (Reddien and Alvarado, 2004; Salo, 2006); and to grow and degrow depending of the environmental conditions and food availability (Baguñá and Romero, 1981). These characteristics are due to the presence of an adult stem cell population – called neoblasts – that is able to give rise to any planarian cell type (Reddien and Alvarado, 2004; Salo, 2006). The high regenerative capacity of planarians, with the presence of a unique totipotent stem cell system, provides an ideal model for studying cell renewal, regeneration, and stem cell regulation. Schmidtea mediterranea is the most common planarian species used in molecular biology to perform molecular and cellular studies, because it presents special features that optimize research. For instances, they are easily maintained as a stable clonal line in the laboratory due to their robust ability to regenerate, which allows a uniform genetic background and minimizes the experimental variability. Here we detail the protocol used in our laboratory to maintain the planarian species Schmidtea mediterranea.

Materials and Reagents

  1. Glass Tupperware (Figure 1B)
    The size may vary depending on the requirements of each experiment. You must avoid large containers that can be too heavy and difficult to manipulate. 
  2. Assexual planarian clonal line of the asexual strain of Schmidtea mediterranea
    This species can be found in coastal areas in the Western Mediterranean. The origin of the strain used in most labs are the fountains of Montjuic in Barcelona. The easiest source nowadays is asking directly from our laboratory or any that has already stablished it as a model organism. 
  3. Organic beef liver
    Note: It can be purchased in any butcher shop that sells organic meat.
  4. NaOH (Merck, catalog number: 106462 )
  5. MgSO4•7H2O (Merck, catalog number: 105886 )
  6. NaHCO3 (Merck, catalog number: 106329 )
  7. KCl (Merck, catalog number: 104936 )
  8. MgCl2•6H2O (Merck, catalog number: 105833 )
  9. CaCl2•2H2O (Merck, catalog number: 102382 )
  10. MilliQ water
  11. 100x Planarians Artificial Medium (PAM) stock solution (see Recipes)
  12. 100x CaCl2 Stock solution (see Recipes)

Equipment

  1. "Planarium"–room or incubator at 20 °C (Figure 1C)
  2. 1 L Beaker
  3. Magnetic stirrer
  4. Stir bar
  5. 1 L Graduated cylinder
  6. 1 L Crystal bottle
  7. Water tank


    Figure 1. Culture of Schmidtea mediterranea. A. In vivo planarian from the species Schmidtea mediterranea. B. Glass Tupperware with planarians. C. “Planarium” (room at 20 °C).

Procedure

  1. Prepare 100x Planarians Artificial Medium (PAM) stock solution (see Recipe 1).
    Note: Separated stocks of 100x PAM water and 100x CaCl2•2H2O solution should be prepared to avoid salt precipitation. It is not recommended to prepare a higher concentrated stock because salts may precipitate. Dilute the appropriate amount of both solutions and mix them just before their use (see Recipes). Do not store the diluted stocks in the water tanks for more than 1 week.
  2. Prepare 100x CaCl2 Stock solution (see Recipe 2).
  3. When required, dilute the stocks to 1x in Millipore water using the water tanks. For 10 L of 1x PAM water use 100 ml of each stock.
  4. Put planarians, obtained from a clonal line strain of Schmidtea mediterranea in a glass Tupperware and fill it with 1x PAM water up to ¾ of the Tupperware capacity (Figure 1B). Keep planarians in a "Planarium": a separated room or incubator with controlled temperature (around 20 °C) and maintained in the dark.
    Note: To increase the population, planarians can be cut at postpharyngeal level, which is the region that fissions during natural asexual reproduction. The fragments will completely regenerate the missing parts in two weeks. If planarians are big (> 0.5 cm) and a high increase in the population is desired, an additional cut can be performed prepharyngeally.
  5. To promote planarians grow and reproduction, feed them 2 or 3 times a week with organic beef liver. To do that, immerse small pieces of liver in the water during 5-7 h (a piece of 4 cm2 cut in smaller fragments can be added in a 150 cm2 Tupperware). After this time, remove the liver and change with the PAM water. When mucus is observed in the Tupperware walls, clean it with a piece of paper without touching planarians and add new 1x PAM water.
    Notes:
    1. For long-term storage, beef liver should be cut into small pieces (around 3-5 cm), wrapped in foil and frozen. When use it to feed planarians, remove the foil and cut it into small pieces with a scalpel.
    2. Be aware to cut some openings in the Tupperware cover to allow the gas exchange.
    3. Normally PAM water must be changed only after feeding. However, when mucus is clearly observed in the Tupperware walls, planarian water should be changed even if they have not been fed. You must be aware to avoid infections.
  6. To increase the planarian population, planarians can be cut at postpharyngeal level, which is the region that fissions during natural asexual reproduction. The fragments will completely regenerate the missing parts in two weeks. If planarians are big (> 0.5 cm) and a high increase in the population is desired, an additional cut can be performed prepharyngeally. The density of the planarians can be varied according to the specific requirements of each researcher. The culture showed in Figure 1B corresponds to a low-density culture. It can be increased up to 2-3 times more.

Data analysis

To perform experiments, planarians of the same size should be chosen to avoid results discrepancy due to differences in size. For the same reason, only fully regenerated planarians should be used (see Figure 2).


Figure 2. Tail regeneration of Schmidtea mediterranea. A. Planarian that has been just cut tail. B. Planarian that has partially regenerated the tail (see white blastema pointed with a yellow arrow), 6-8 days after the cut. C. Fully regenerated planarian, 10-13 days after the cut.

Recipes

  1. 100x Planarians Artificial Medium (PAM) stock solution
    1.6 mM NaCl
    1.0 mM MgSO4•7H2O
    1.2 mM NaHCO3
    0.1 mM KCl
    0.1 mM MgCl2•6H2O
    1. Add around 750 ml of MilliQ water to a beaker
    2. Put the beaker in the magnetic stirrer with a stir bar
    3. Add the salts one by one to the MilliQ water to reach the appropriated concentration. Before adding the next salt, confirm that the previous one is totally dissolved
    4. Transfer the solution to 1 L graduated cylinder and adjust the volume to 1 L
    5. Store this stock in a 1 L glass bottle in the “Planarium”
  2. 100x CaCl2 Stock solution
    1.0 mM CaCl2•2H2O
    1. Add around 750 ml of MilliQ water to a beaker
    2. Put the beaker in the magnetic stirrer with a stir bar
    3. Add the salt to the MilliQ water to reach the appropriated concentration
    4. Transfer the solution to 1 L graduated cylinder and adjust the volume to 1 L
    5. Store this stock in a 1 L glass bottle in the “Planarium” 
    Note: If stock solutions precipitate, agitate them for a while in a magnetic stirrer and then filtrate the solutions.
  3. PAM water
    0.016 mM NaCl
    0.01 mM MgSO4•7H2O
    0.012 mM NaHCO3
    0.001 mM KCl
    0.001 mM MgCl2•6H2O
    0.01 mM CaCl2•2H2O
    Note: PAM water must be prepared from two different 100x stock solutions (see Recipes 1 and 2), since the CaCl2 must be added only when required, to avoid precipitation.

Acknowledgments

We acknowledge Eudald Pascual for the images in Figure 2. This work was supported by grant BFU2008-01544 and BFU2014-56055-P (Ministerio de Educación y Ciencia) and grant 2009SGR1018 (AGAUR). N.S. was supported by the APIF fellowship from the Universitat de Barcelona. This work is adapted from previous literature (Cebrià and Newmark, 2005).

Competing interests

The authors declare no conflicts of interest or competing interests.

References

  1. Baguñá, J. and Romero, R. (1981). Quantitative analysis of cell types during growth, degrowth and regeneration in the planarians Dugesia mediterranea and Dugesia tigrina. Hydrobiologia 84(1): 181-194.
  2. Cebrià, F. and Newmark, P. A. (2005). Planarian homologs of netrin and netrin receptor are required for proper regeneration of the central nervous system and the maintenance of nervous system architecture. Development 132(16): 3691-3703.
  3. Reddien, P. W. and Alvarado, A. S. (2004). Fundamentals of planarian regeneration. Annu Rev Cell Dev Biol 20: 725-757.
  4. Salo, E. (2006). The power of regeneration and the stem-cell kingdom: freshwater planarians (Platyhelminthes). Bioessays 28(5): 546-559.

简介

在过去的几年中,涡虫已成为研究再生和干细胞生物学的独特模型动物。 虽然它们具有显着的再生能力,但很长一段时间以来,人们已经建立了理解涡虫干细胞生物学及其再生过程基础的分子工具。 这种推动是由于测序基因组的可用性以及干扰RNA和新一代测序等新技术的发展,这些技术促进了分子和基因水平的涡虫再生研究。 由于这些原因,在实验室中保持健康稳定的涡虫种群对于进行可重复的实验至关重要。 在这里,我们详细介绍了我们实验室使用的协议,以维护最广泛作为模型的涡虫种类 Schmidtea mediterranea 。
【背景】涡虫是双侧对称的扁形动物,是超级寄主lophotroczoa的成员。有陆地,海洋和淡水涡虫。它们主要捕食受伤的昆虫,昆虫幼虫和其他无脊椎动物。涡虫是缺乏循环系统,骨骼系统和呼吸系统的三倍体和动脉粥样硬化动物(图1A)。这些动物在几天内截肢后具有恢复身体任何缺失部位的惊人能力(Reddien和Alvarado,2004; Salo,2006);并根据环境条件和食物供应情况进行生长和驯化(Baguñá和Romero,1981)。这些特征是由于存在成体干细胞群 - 称为新生细胞 - 能够产生任何涡虫细胞类型(Reddien和Alvarado,2004; Salo,2006)。涡虫的高再生能力,具有独特的全能干细胞系统,为研究细胞更新,再生和干细胞调节提供了理想的模型。 Schmidtea mediterranea 是分子生物学中用于进行分子和细胞研究的最常见的涡虫种类,因为它具有优化研究的特殊功能。例如,由于其强大的再生能力,它们很容易在实验室中作为稳定的克隆系维持,这允许均匀的遗传背景并最小化实验变异性。在这里,我们详细介绍了我们实验室用于维护涡虫种类 Schmidtea mediterranea 的方案。

关键字:真涡虫, 真涡虫, 培养, 真涡虫, 模式生物, 再生

材料和试剂

  1. Glass Tupperware(图1B)
    尺寸可根据每个实验的要求而变化。您必须避免使用太重且难以操作的大型容器。 
  2. Schmidtea mediterranea 该物种可以在西地中海的沿海地区找到。大多数实验室使用的菌株的起源是巴塞罗那Montjuic的喷泉。现在最简单的来源是直接从我们的实验室或任何已经建立它作为模型生物的人那里询问。 
  3. 有机牛肝
    注意:可以在任何出售有机肉的肉店购买。
  4. NaOH(默克,目录号:106462)
  5. MgSO 4 •7H 2 O(默克,目录号:105886)
  6. NaHCO 3 (默克,目录号:106329)
  7. KCl(默克,目录号:104936)
  8. MgCl 2 •6H 2 O(默克,目录号:105833)
  9. CaCl 2 •2H 2 O(默克,目录号:102382)
  10. MilliQ水
  11. 100x Planarians人工培养基(PAM)储备液(见食谱)
  12. 100x CaCl 2 储备溶液(见食谱)

设备

  1. “Planarium” - 室温或20°C的培养箱(图1C)
  2. 1升烧杯
  3. 电磁搅拌器
  4. 搅拌棒
  5. 1 L量筒
  6. 1升水晶瓶
  7. 水箱


    图1.培养 Schmidtea mediterranea 。 A. 体内来自物种 Schmidtea mediterranea 。 B.带有涡虫的玻璃特百惠。 C.“Planarium”(20°C的房间)。

程序

  1. 准备100x Planarians人工培养基(PAM)储备液(见食谱1)。
    注意:100x PAM水和100x CaCl的分离库存 2 •2H 2 应准备好溶液以避免盐析。不建议制备更高浓度的原料,因为盐可能会沉淀。稀释适量的两种溶液,并在使用前混合(参见食谱)。不要将稀释后的原料储存在水箱中超过1周。
  2. 准备100x CaCl 2 储备液(参见配方2)。
  3. 需要时,使用水箱将原料稀释至Millipore水中1倍。对于10L 1x PAM水,使用100ml每种原料。
  4. 将一种来自 Schmidtea mediterranea 的克隆系菌株的涡虫放入玻璃特百惠中,并用1x PAM水填充至Tupperware容量的3/4(图1B)。将涡虫放在“Planarium”中:一个分开的房间或孵化器,温度可控(约20°C)并保持在黑暗中。
    注意:为了增加种群,可以在咽后水平切割涡虫,这是在自然无性繁殖期间裂变的区域。片段将在两周内完全再生缺失的部分。如果涡虫大(> 0.5厘米)并且需要大量增加,可以预先进行额外的切割。
  5. 为了促进涡虫的生长和繁殖,每周喂食有机牛肝2至3次。为此,在5-7小时内将小块肝脏浸入水中(一块4厘米 2 切成较小的碎片可以在150厘米 2 中加入特百惠)。在此之后,取出肝脏并用PAM水改变。当在特百惠墙壁上观察到粘液时,用一张纸清洁它而不接触涡虫并添加新的1x PAM水。
    注意:
    1. 为了长期储存,应将牛肝切成小块(约3-5厘米),用铝箔包裹并冷冻。当用它来喂食涡虫时,取出金属箔并用手术刀将其切成小块。
    2. 请注意在特百惠盖板上切开一些开口,以便进行气体交换。
    3. 通常只有喂食后才能更换PAM水。然而,当在特百惠墙壁上清楚地观察到粘液时,即使没有喂食,也应改变涡虫水。你必须意识到要避免感染。
  6. 为了增加涡虫种群,可以在咽后水平切割涡虫,这是在自然无性繁殖期间裂变的区域。片段将在两周内完全再生缺失的部分。如果涡虫大(> 0.5cm)并且需要人口的高增加,则可以预先进行额外的切割。涡虫的密度可以根据每个研究人员的具体要求而变化。图1B中显示的培养物对应于低密度培养物。它可以增加2-3倍。

数据分析

为了进行实验,应选择相同大小的涡虫,以避免由于大小差异导致的结果差异。出于同样的原因,只应使用完全再生的涡虫(见图2)。


图2. Schmidtea mediterranea的尾部再生 。 A.刚刚切断尾巴的Planarian。 B.在切割后6-8天,使尾部部分再生的Planari(参见用黄色箭头指向的白色blastema)。 C.完全再生的涡虫,切割后10-13天。

食谱

  1. 100x Planarians人工中(PAM)原液解决方案
    1.6 mM NaCl
    1.0mM MgSO 4 ·7H 2 O
    1.2 mM NaHCO 3
    0.1 mM KCl
    0.1mM MgCl 2 •6H 2 O
    1. 将约750ml MilliQ水加入烧杯中
    2. 用搅拌棒将烧杯放入磁力搅拌器中
    3. 将盐逐一加入MilliQ水中,达到适当的浓度。在添加下一个盐之前,确认前一个盐已完全溶解
    4. 将溶液转移至1 L量筒并将体积调节至1 L.
    5. 将此库存储存在“Planarium”的1升玻璃瓶中
  2. 100x CaCl 2 库存溶液
    1.0 mM CaCl 2 •2H 2 O
    1. 将约750ml MilliQ水加入烧杯中
    2. 用搅拌棒将烧杯放入磁力搅拌器中
    3. 将盐加入MilliQ水中以达到适当的浓度
    4. 将溶液转移至1 L量筒并将体积调节至1 L.
    5. 将此库存储存在“Planarium”中的1升玻璃瓶中 
    注意:如果储备溶液沉淀,在磁力搅拌器中搅拌一会儿,然后过滤溶液。
  3. PAM水
    0.016 mM NaCl
    0.01mM MgSO 4 ·7H 2 O
    0.012 mM NaHCO 3
    0.001 mM KCl
    0.001 mM MgCl 2 •6H 2 O
    0.01 mM CaCl 2 •2H 2 O
    注意:PAM水必须从两种不同的100x储备溶液中制备(参见配方1和2),因为CaCl 2 必须只在需要时添加,以避免降水。

致谢

我们对图2中的图像感谢Eudald Pascual。这项工作得到了补助金BFU2008-01544和BFU2014-56055-P(MinisteriodeEducaciónyCiencia)和2009SGR1018(AGAUR)的资助。 N.S。得到了巴塞罗那大学APIF奖学金的支持。这项工作改编自以前的文献(Cebrià和Newmark,2005)。

利益争夺

作者声明没有利益冲突或竞争利益。

参考

  1. Baguñá,J。和Romero,R。(1981)。 对涡虫中的生长,去除和再生过程中细胞类型的定量分析 Dugesia mediterranea 和 Dugesia tigrina 。 Hydrobiologia 84(1):181-194。
  2. Cebrià,F。和Newmark,P。A.(2005)。 netrin 和 netrin receptor 的Planarian同系物是正确再生中枢神经系统和维持神经系统结构所必需的。 发展 132(16):3691-3703。
  3. Reddien,P.W。和Alvarado,A。S.(2004)。 涡虫再生的基础知识。 Annu Rev Cell Dev Biol 20:725-757。
  4. Salo,E。(2006)。 再生和干细胞王国的力量:淡水涡虫(Platyhelminthes)。 Bioessays 28(5):546-559。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Sousa, N. D. and Adell, T. (2018). Maintenance of Schmidtea mediterranea in the Laboratory. Bio-protocol 8(19): e3040. DOI: 10.21769/BioProtoc.3040.
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