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Jun 2018

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Acute Isolation of Cells from Murine Sino-atrial Node
小鼠窦房结细胞的快速分离   

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Abstract

The cardiac conduction system allows the synchronized propagation of electrical activity through heart muscle. This is initiated by the spontaneous activity of the specialized pacemaker cells of the sino-atrial node (SAN). The SAN region underlies automaticity in mammals and therefore has a crucial role in the pathogenesis of cardiac disorders such as arrhythmia. Isolation of SAN tissue and SAN cells is critical to advance our understanding of SAN structure and function in health and disease. Initially, isolation of SAN tissue and SAN cells was carried out in the rabbit owing to its larger size and similar electrical properties to human. This protocol was optimized by Mangoni and Nargeot (2001) for use in mice to take advantage of advancements in transgenic models. Here, we provide a step-by-step guide to dissecting the SAN tissue and isolating pacemaker cardiomyocytes from mouse hearts using an enzyme digestion approach.

Keywords: Conduction system (传导系统), SAN (SAN), Mouse SAN cells (小鼠SAN细胞), Primary cell isolation (原代细胞分离), Pacemaker cells (起搏细胞)

Background

Cardiovascular diseases and abnormalities are a major cause of morbidity and mortality and are major burden on healthcare systems. Arrhythmias as result of changes in the structure, rate and rhythm of the cardiac conduction system underlie many cardiac disorders. In the past, a lack of availability of homogenous conduction system cell lines has proven to be a hindrance to investigators wishing to study the function of conduction system cells in health and disease. The emergence of transgenic animal models of cardiac abnormalities provides an even greater impetus to reliably isolate healthy conduction system cells. Intact nodes allow investigations into expression profiles of important signaling proteins and genes using immunohistochemistry and qPCR approaches. More detailed functional analysis is possible on isolated single nodal cells using patch-clamp for example. The SAN tissue and the cells it consists of were first isolated many decades ago (Noble and Tsien, 1968). However, the cell population isolated was generally a mixed population. SAN specific cells were first isolated from rabbit SAN by DiFranscesco et al. (1986) in the mid-1980s to allow study of ion channel expression. More recently, the protocol was adapted for isolation of SAN cells from mice by Mangoni and Nargeot (2001). This has allowed transgenic mouse models specific to the SAN to be studied in greater detail. We describe the procedure for isolating the SAN tissue and single SAN cells. The intact node can be used for investigation of gene and protein expression as well as for tissue histology, whilst acutely isolated cells can be interrogated in detail for their electrophysiological properties.

Materials and Reagents

  1. 15 ml Falcon tubes (Sarstedt, catalog number: 65.554.001)
  2. Petri Dish 100 x 21 mm (Thermo Fisher Scientific, NuncTM, catalog number: 172931)
  3. Sylgard 170-Silicone Elastomer (Dow Corning, Farnell, catalog number: 101693)
  4. 10 ml Glass vials with snap cap (VWR international, catalog number: 548-0555)
  5. 1 ml Syringe (Terumo, Scientific laboratory supplies, catalog number: SYR6200)
  6. 26 G needle (VWR International, catalog number: 613-3896)
  7. Insect pins (Fine Science Tools, catalog number: 260002-13)
  8. Pyrex disposable borosilicate culture tubes, Corning 99445-10 (Thermo Fisher Scientific, catalog number: 13023073)
  9. Multi-purpose parafilm M (Sigma-Aldrich, catalog number: P6543)
  10. Glass or plastic Beaker, 100 ml and 200 ml (Laboratory specific)
  11. Borosilicate Glass Pasteur Pipettes, autoclaved (Fisher Scientific, catalog number: 13-678-20C)
  12. 13 mm glass coverslips (VWR, catalog number: 631-0148)
  13. 12-well cell culture plates, Cellstar (Greiner Bio-One, catalog number: 665 180)
  14. Adult mice, 6-12 weeks, e.g., C57/Bl6 (The Jackson Laboratory, catalog number: 000664)
  15. Bovine Serum Albumin (Sigma-Aldrich, catalog number: A6003)
  16. Collagenase Type II (Worthington Biochemical Corp, catalog number: 4176)
  17. Elastase (Worthington Biochemical Corp, catalog number: 2292)
  18. Protease XIV (Sigma-Aldrich, catalog number: P5147)
  19. Laminin (Sigma-Aldrich, catalog number: L2020)
  20. Deionized, filtered water (dH2O) (Merck Millipore, Milli-Q)
  21. Sodium Chloride (NaCl) (Sigma-Aldrich, catalog number: S7653)
  22. Potassium Chloride (KCl) (Sigma-Aldrich, catalog number: P9333)
  23. Potassium Phosphate (KH2PO4) (Sigma-Aldrich, catalog number: P0662)
  24. L-Glutamic Acid (C5H9NO4) (Sigma-Aldrich, catalog number: 09581)
  25. Potassium Glutamate (C5H8KNO4·H2O) (Sigma-Aldrich, catalog number: G1501)
  26. Potassium Hydroxide pellets (KOH) (Sigma-Aldrich, catalog number: P6310)
  27. Sodium Hydroxide pellets (NaOH) (Thermo Fisher Scientific, catalog number: S/4920/53)
  28. HEPES-NaOH (Sigma-Aldrich, catalog number: H7006)
  29. HEPES-KOH (Sigma-Aldrich, catalog number: H0527)
  30. D-Glucose (Sigma-Aldrich, catalog number: G5767)
  31. Taurine (Sigma-Aldrich, catalog number: T8691)
  32. Calcium Chloride (CaCl2) 1 M solution (Fluka, catalog number: 21114)
  33. Magnesium Chloride (MgCl2) 1 M solution (Fluka, catalog number: 63026)
  34. DL-β-Hydroxybutyric acid sodium salt (Sigma-Aldrich, catalog number: H6501)
  35. Ketamine (Narketen-10) (Vetoquinox, procured via biological services unit)
  36. Xylazine (Rompun 2% w/v) (Bayer, procured via biological services unit)
  37. Heparin Sodium Salt (1,000 U/ml) (Wockhardt, procured via biological services unit)
  38. Tyrode solution (see Recipes)
  39. Tyrode-BSA solution (see Recipes)
  40. Low Ca2+/Mg2+ solution (see Recipes)
  41. Kraftbruhe (KB) solution (see Recipes)
  42. SAN storage solution (see Recipes)
  43. Enzyme digestion solution (see Recipes)
  44. Re-adaptation solution (see Recipes)
  45. Storage solution (mM) (see Recipes)
  46. Heparin/Ketamine/Xylazine Injection (see Recipes)
  47. Sylgard dissection dishes (see Recipes)

Equipment

  1. Scale and weighing (Laboratory specific)
  2. Dissecting scissors (World Precision Instruments, catalog number: 14192)
  3. Student Vannas scissors (World Precision Instruments, catalog number: 501777)
  4. Fine Vannas Scissors (World Precision Instruments, catalog number: 500086)
  5. Fine Vannas Scissors curved (World Precision Instruments, catalog number: 501232)
  6. 2x Dumont No.5 forceps (World Precision Instruments, catalog number: 500085)
  7. Zeiss Stereomicroscope Stemi2000 (Zeiss, catalog number: 455052)
  8. Grant Water Bath (Scientific Laboratory Supplies, catalog number: BAT3310)
  9. Stuart SSL4 See-Saw Rocker (Scientific Laboratory Supplies, catalog number: MIX2066)
  10. Cell Culture Incubator (Laboratory-specific)
  11. PIPETMAN Classic P10 (Gilson, catalog number: F144802)
  12. PIPETMAN Classic P100 (Gilson, catalog number: F144802)
  13. PIPETMAN Classic P200 (Gilson, catalog number: F144802)
  14. PIPETMAN Classic P1000 (Gilson, catalog number: F144802)
  15. Centrifuge 5810 R swing bucket for conical tubes (Eppendorf, model: 5810 R)
  16. Diamond cutting tool for glass (Amazon)
  17. Culture hood

Procedure

The procedure is illustrated in Figure 1.
Note: Stock solutions for dissection and isolation of the SAN can be prepared the day before isolation (Recipes 1, 2, 4, 5 and 7) as it can take ~1 h to make them. Solutions should be kept in the fridge (~4-8 °C) and can be used for isolation for up to a week. For working solutions, such as the enzyme solution it is advisable to make on the day. All working solutions need to be pre-warmed (in a water bath) at 37 °C before use (~15-20 min). It is critical that CaCl2 (10 µl of 100 mM stock to 2.5 ml-final concentration 400 µM) is added to the enzyme solution, otherwise tissue digestion will be compromised.


Figure 1. Schematic of the protocol for the isolation of SAN cells from a mouse heart


  1. Preparation
    1. Before dissection of the SAN, prepare the following Pyrex glass tubes:
      1. 2 x 2.5 ml of low Ca2+/Mg2+ solution (Recipe 2);
      2. 1 x 2.5 ml of enzyme solution (Recipe 3 and Note above);
      3. 4 x 2.5 ml of KB solution (Recipe 4).
    2. Beaker with pre-warmed Tyrode solution (Recipe 1).
    3. 2 dissection dishes with pre-warmed Tyrode solution.
    4. Heparin/Xylazine/Ketamine injection (Recipe 8).
    5. Assemble tools required for dissection (dissecting scissors, fine forceps, Vannas scissors).
    6. Prepare a 12-well plate with laminin-coated coverslips. Coat coverslips with 1 µl of Laminin per coverslip and spread using a P200 tip and allow to dry in the culture hood with the lid off.

  2. Heart removal from chest cavity
    1. Anesthetize mice with an intraperitoneal injection of Xylazine/Ketamine/Heparin mix (adhere to Institute guidelines).
    2. Lay the mouse in the supine position (Figure 2) and spray the chest area with 70% ethanol. Open the chest cavity by cutting through the rib cage using dissecting scissors and remove the intact heart by holding it at the aortic arch with forceps and cutting just above the forceps using the student Vannas spring scissors. Do not worry about taking extra-cardiac tissue as this can be cleaned later. Rinse the heart in a beaker filled with Tyrode solution. Be careful not to cut/damage atrial appendage regions of the heart.
    3. Place the heart, pinned at the apex with an insect pin on a dissection dish submerged with Tyrode solution. The heart should be placed as it would be in the mouse chest cavity (see Figure 2). Remove non-cardiac tissue such as lungs, thymus, connective tissue and fat under a microscope. Make a small incision in the ventricles using Vannas scissors and flush the heart of blood using a Pasteur pipette filled with Tyrode solution. Make sure to avoid air bubbles.
    Note: The mouse can be euthanized by cervical dislocation without anesthesia if one is careful not to crush the heart tissue. Heparin will prevent blood clots and make dissection/cleaning of the node easier. Tyrode solution used to rinse the heart can be at RT or pre-warmed–we found no difference in the quality of tissue isolated between the two conditions.


    Figure 2. Dissection of the mouse heart and isolation of the SAN. A. The anesthetized mouse is placed in the supine position and incisions made where indicated (dotted lines) to access the cardiac cavity. B. Removal of the rib cage shows the position of the heart. The heart is dissected by cutting at the aortic arch. The lungs can be removed before or after excision. C. Excised heart showing the right and left atrial appendage (RA and LA), right and left ventricles (RV and LV) and the aorta (Ao). D. The heart is pinned to the dissection dish- the dotted line shows where to cut to separate the atrial/nodal tissue from the ventricles without damaging the SAN. E. Intact SAN, RA and LA after separation from the ventricles. The incision seen on the left ventricle was used to flush out the blood from the heart before separation of the upper chambers from the ventricles. F and G. The SAN (within dotted area) and surrounding tissue after the fat and connective tissue have been removed. CT, cristae terminalis. IAS, intra-atrial septum.

  3. SAN dissection
    1. Under the microscope, carefully make a horizontal incision using students Vannas scissors at the junction between the ventricles and atria (see Figure 2). The ventricles should stop beating and can be discarded. The upper chambers with the atrial appendages and conduction tissue should continue to beat.
    2. Flip the atrial/conduction tissue so that the aorta is facing you with the right atria on your left side.
    3. Using insect pins carefully pin the atrial/conduction tissue in a clean Sylgard dissection dish (Recipe 9) containing Tyrode solution (see Figure 2). Place pins strategically on the left and right atria, on the edges of the aorta (see Figure 2) or superior vena cava and inferior vena cava (if present). Do not over-stretch the tissue. Carefully remove the remaining ventricular tissue, if necessary, using fine Vannas scissors.
    4. Once the tissue is free of ventricular muscle, cut open the right atrial appendage pocket and re-pin to reveal the Cristae terminalis (CT). The SAN (transparent) will be clearly visible between the inter-atrial septum and the cristae terminalis (see Figure 2).
    5. Carefully remove any fat/connective tissue from beneath the SAN.
    6. Finally, carefully cut the SAN with fine Vannas scissors and, transfer to a Pyrex tube containing pre-warmed low Ca2+/Mg2+ solution with fine forceps.
    Note: The superior and inferior vena cava are not always seen in every preparation, however the aorta should be visible and is ideal to pin at the top. The bottom end can be easily pinned using connective tissue or part of the right and left atrial appendages. When cleaning the tissue use sharp forceps (with no hooks) and curved Vannas scissors, these allow you to cut away from the tissue and prevent damage to the node. It is especially important to be careful when cleaning the underside of the nodal tissue. To do this as thoroughly as possible it may be necessary to unpin the bottom end (or top end) and flip to allow easier access. The node will generally continue to beat for a prolonged period of time (~10-20 min)–this can be important when dissecting for the first few attempts because it allows visualization of the area of interest. If the node stops beating, it can be restarted by gently pipetting fresh Tyrode solution on to it.

  4. Enzyme digestion of SAN tissue (see the schematic Figure 1)
    All solutions should be pre-warmed at 37 °C in a water bath.
    1. Place the SAN tissue into a pre-prepared Pyrex tube containing low Ca2+/Mg2+ solution. Incubate for 2 min at 37 °C (in the water bath). Using a fire-polished Pasteur pipette carefully transfer the tissue in to the second low Ca2+/Mg2+ tube for a further 4 min.
    2. Transfer the SAN tissue in to the enzyme solution for 10-15 min at 37 °C. Agitate manually every 2-3 min with a fire polished Pasteur pipette. The tissue should start to look less structurally rigid.
    3. Wash the tissue 4 times in the KB solution. The first 3 washes should be for 2 min followed by a fourth and final wash for 4 min.
    4. Transfer the tissue and KB solution from the final wash to a 5 or 10 ml glass vial. Gently triturate (pipette up and down) the tissue with an appropriately sized (see Notes) fire-polished glass Pasteur pipette for ~30-60 s, avoiding air bubbles. Allow the cell suspension to rest at room temperature for 5 min.

  5. Re-adaptation of SAN cells
    1. Transfer the triturated cell suspension to a 15 ml Falcon tube.
    2. Cumulatively add the following volumes of the NaCl/CaCl2 solution (Recipe 5) to the Falcon tube containing the cell suspension and place on the rocking platform.
      25 µl for 4 min
      50 µl for 4 min
      70 µl for 4 min
      90 µl for 4 min
    3. Add 360 µl of Tyrode-BSA (Recipe 6) to the cell suspension and place on the rocker for 4 min.
    4. Centrifuge the cell suspension at ~60-70 x g for 7 min. There should be a small pellet of cells.
    5. Carefully remove 2-2.2 ml of the supernatant using a 1 ml Gilson Pipetman (~0.8-1 ml supernatant should remain).
    6. Gently re-suspend the pellet of cells in the remaining supernatant using 1 ml Gilson Pipetman and place roughly an equal amount (~60-80 µl) on the pre-prepared laminin coated coverslip (within a 12-well culture plate). Incubate the plate at 37 °C (95% O2/5% CO2) cell culture incubator for ~30-45 min to allow cells to adhere to the coverslips.
    7. Check cells under the microscope (using the 20x objective) to see if cells are viable–cells should. Add 1 ml/well of pre-warmed storage solution (Recipe 7) or Tyrode-BSA. Cells are now ready to use.
      Morphologically SAN cells consist of 3 types: Spindle-shaped, spider-like and elongated (see Figure 3).
      Note: We found that placing the cells in Tyrode-BSA was just as effective as placing them in storage solution. The number of total cells can be very variable depending on the success of the preparation, however on average we expect to see ~100-120 (5-10 per coverslip) viable cells per preparation.


      Figure 3. Morphologies of the three types of acutely isolated single SAN cells from mouse heart. A. Spindle-shaped. B. Spider-like. These cells have 1 or more processes. C. Elongated spindle-shaped. Healthy cells beat spontaneously. Scale bar: 20 µm.

Recipes

  1. Tyrode solution
    140 mM NaCl
    5.4 mM KCI
    1.2 mM KH2PO4
    1.8 mM CaCI2
    1.2 mM MgCI2
    5 mM HEPES-NaOH
    5.5 mM D-Glucose
    Adjust the pH to 7.4 with NaOH or HCI if pH basic
  2. Low ‘Ca2+/Mg2+’ solution
    140 mM NaCl
    5.4 mM KCI
    1.2 mM KH2PO4
    0.2 mM CaCI2
    0.5 mM MgCI2
    50 mM Taurine
    5 mM HEPES-NaOH
    5.5 mM D-Glucose
    1 mg/ml BSA
    Adjust the pH to 6.9 with NaOH or HCI if pH if basic
  3. Enzyme solution (made in 2.5 ml of low Ca2+/Mg2+ solution [Recipe 2])
    1 mg/ml Collagenase Type II
    1 mg/ml Protease XIV
    0.5 mg/ml Elastase
    10 μl of 100 mM CaCl2 (400 µM final concentration)
  4. Kraftsbuhe ‘KB’ medium
    70 mM L-glutamic acid
    20 mM KCI
    80 mM KOH
    10 mM KH2PO4
    10 mM Taurine
    10 mM HEPES-KOH
    1 mg/ml BSA
    Adjust the pH to 7.4 with KOH
  5. Re-adaptation solution
    10 mM NaCl
    1.8 mM CaCI2 
  6. Tyrode-BSA
    Tyrode solution (Recipe 1)
    1 mg/ml BSA 
  7. Storage solution
    100 mM NaCl
    35 mM KCI
    4 mM KH2PO4
    1.3 mM CaCI2
    0.7 mM MgCI2
    2 mM Taurine
    14 mM Potassium glutamate
    0.2 mM (±) D-β-butyric acid
    1 mg/ml BSA
    Adjust the pH to 7.4 with NaOH
  8. Heparin/Ketamine/Xylazine Injection
    1. Heparin
      Dilute Heparin stock (e.g., 1,000 IU/ml) to 200 IU/ml in sterile saline–e.g., 1 ml of Heparin in 4 ml of saline. This solution can be kept in the fridge for up to 4 weeks. Use ~1 IU/g (mouse weight). For example, 100 µl of 200 IU/ml heparin for a 20 g mouse
    2. Ketamine
      Use 0.5 mg/g. For example, 100 µl of 100 mg/ml stock for 20 g mouse
    3. Xylazine
      Use 0.1 mg/g. For example, 100 µl of 20 mg/ml stock for 20 g mouse
    4. For injection make a cocktail of heparin/ketamine/xylazine of between 300 µl to 500 µl per mouse
  9. Sylgard dissection dishes
    1. Dissection dishes were made using Sylgard 170-Silicone Elastomer (Dow Corning) as per manufacturer’s instructions
    2. Parts A and B were mixed in equal amounts in a Petri Dish (100 x 21 mm). The amounts used are dependent on the thickness required (we recommend enough for 50% depth)
    3. The Sylgard mix allowed to set at 37 °C for at least 24 h (this can be done at room temperature but it will take longer to set)

Acknowledgments

We would like to thank the BHF for funding (RG/15/15/311742). The work was facilitated by the NIHR Biomedical Research Centre at Barts. Special thanks to Dr Mangoni for allowing Dr Nobles to visit his laboratory to learn the procedure for isolation of murine SAN cardiomyocytes. This protocol was adapted/modified from the protocol of Mangoni and Nargeot (2001).

Competing interests

We have no conflicts of interest.

Ethics

All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the British Home Office regulations (covered by project license PE9055EAD) and by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

References

  1. DiFrancesco, D., Ferroni, A., Mazzanti, M. and Tromba, C. (1986). Properties of the hyperpolarizing-activated current (if) in cells isolated from the rabbit sino-atrial node. J Physiol 377: 61-88.
  2. Mangoni, M. E. and Nargeot, J. (2001). Properties of the hyperpolarization-activated current (I(f)) in isolated mouse sino-atrial cells. Cardiovasc Res 52(1): 51-64.
  3. Noble, D. and Tsien, R. W. (1968). The kinetics and rectifier properties of the slow potassium current in cardiac Purkinje fibres. J Physiol 195(1): 185-214.

简介

心脏传导系统允许电活动通过心肌同步传播。这是由窦房结(SAN)的专用起搏器细胞的自发活动引起的。SAN区域是哺乳动物自动化的基础,因此在诸如心律不齐等心脏疾病的发病机理中具有至关重要的作用。隔离SAN组织和SAN细胞对于增进我们对健康和疾病中SAN结构和功能的理解至关重要。最初,由于兔子的体形较大且与人的电特性相似,因此在兔子中进行了SAN组织和SAN细胞的分离。该方案由Mangoni和Nargeot(2001)优化,用于小鼠,以利用转基因模型的先进性。在这里,我们提供了使用酶消化方法解剖SAN组织并从小鼠心脏中分离起搏器心肌细胞的分步指南。
【背景】心血管疾病和异常是发病和死亡的主要原因,也是医疗保健系统的主要负担。心律失常是心脏传导系统结构,速率和节律变化的结果,是许多心脏疾病的基础。过去,事实证明缺乏同质的传导系统细胞系是希望研究传导系统细胞在健康和疾病中作用的研究人员的障碍。心脏异常的转基因动物模型的出现为可靠地分离健康的传导系统细胞提供了更大的动力。完整的结节允许使用免疫组织化学和qPCR方法研究重要信号蛋白和基因的表达谱。例如,可以使用膜片钳对分离的单个结节细胞进行更详细的功能分析。SAN组织及其组成的细胞是几十年前首次分离出来的(Noble和Tsien,1968)。但是,分离的细胞群体通常是混合群体。SAN特异细胞首先是由DiFranscesco等(1986)从兔子SAN中分离出来的,在1980年代中期开始进行离子通道表达的研究。最近,Mangoni和Nargeot(2001)将该协议修改为从小鼠中分离SAN细胞的方法。这使得可以对SAN特有的转基因小鼠模型进行更详细的研究。我们描述了隔离SAN组织和单个SAN细胞的过程。完整的结节可用于研究基因和蛋白质表达以及组织的组织学,而急性分离的细胞可因其电生理特性而被详细询问。

关键字:传导系统, SAN, 小鼠SAN细胞, 原代细胞分离, 起搏细胞

材料和试剂

  1. 15 ml猎鹰管(Sarstedt,目录号:65.554.001)
  2. 培养皿100 x 21毫米(Thermo Fisher Scientific,Nunc TM ,货号:172931)
  3. Sylgard 170-硅酮弹性体(道康宁公司,法内尔,目录号:101693)
  4. 10 ml带卡口盖的玻璃小瓶(VWR国际,目录号:548-0555)
  5. 1毫升注射器(Terumo,科学实验室用品,目录号:SYR6200)
  6. 26 G针(VWR International,目录号:613-3896)
  7. 昆虫针(精细科学工具,目录号:260002-13)
  8. Pyrex一次性硼硅酸盐培养管,Corning 99445-10(Thermo Fisher Scientific,目录号:13023073)
  9. 多功能封口膜M(Sigma-Aldrich,目录号:P6543)
  10. 玻璃或塑料烧杯,分别为100 ml和200 ml(实验室专用)
  11. 高压灭菌的硼硅玻璃巴斯德吸管(Fisher Scientific,目录号:13-678-20C)
  12. 13毫米玻璃盖玻片(VWR,目录号:631-0148)
  13. 12孔细胞培养板,Cellstar(Greiner Bio-One,目录号:665 180)
  14. 成年小鼠,6-12周, eg ,C57 / Bl6(杰克逊实验室,目录号:000664)
  15. 牛血清白蛋白(Sigma-Aldrich,目录号:A6003)
  16. II型胶原酶(沃辛顿生物化学公司,目录号:4176)
  17. 弹性蛋白酶(沃辛顿生物化学公司,目录号:2292)
  18. 蛋白酶XIV(Sigma-Aldrich,目录号:P5147)
  19. 层粘连蛋白(Sigma-Aldrich,目录号:L2020)
  20. 去离子过滤水(dH 2 O)(Merck Millipore,Milli-Q)
  21. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  22. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9333)
  23. 磷酸钾(KH 2 PO 4 )(Sigma-Aldrich,目录号:P0662)
  24. L-谷氨酸(C 5 H 9 NO 4 )(Sigma-Aldrich,目录号:09581)
  25. 谷氨酸钾(C 5 H 8 KNO 4 ·H 2 O)(Sigma-Aldrich,目录号: G1501)
  26. 氢氧化钾颗粒(KOH)(Sigma-Aldrich,目录号:P6310)
  27. 氢氧化钠颗粒(NaOH)(Thermo Fisher Scientific,目录号:S / 4920/53)
  28. HEPES-NaOH(Sigma-Aldrich,目录号:H7006)
  29. HEPES-KOH(Sigma-Aldrich,目录号:H0527)
  30. D-葡萄糖(Sigma-Aldrich,目录号:G5767)
  31. 牛磺酸(Sigma-Aldrich,目录号:T8691)
  32. 氯化钙(CaCl 2 )1 M溶液(Fluka,目录号:21114)
  33. 氯化镁(MgCl 2 )1 M溶液(Fluka,目录号:63026)
  34. DL-β-羟基丁酸钠盐(Sigma-Aldrich,目录号:H6501)
  35. 氯胺酮(Narketen-10)(Vetoquinox,通过生物服务部门采购)
  36. 赛拉嗪(Rompun 2%w / v)(Bayer,通过生物服务部门采购)
  37. 肝素钠盐(1,000 U / ml)(Wockhardt,通过生物服务部门采购)
  38. 提洛德解决方案(请参阅食谱)
  39. Tyrode-BSA解决方案(请参阅食谱)
  40. 低Ca 2 + / Mg 2 + 解决方案(请参阅食谱)
  41. Kraftbruhe(KB)解决方案(请参阅食谱)
  42. SAN存储解决方案(请参阅食谱)
  43. 酶消化液(请参阅食谱)
  44. 重新适应解决方案(请参阅食谱)
  45. 存储解决方案(mM)(请参阅食谱)
  46. 肝素/氯胺酮/甲苯噻嗪注射液(请参阅食谱)
  47. Sylgard解剖盘(请参阅食谱)

设备

  1. 秤和称重(实验室专用)
  2. 解剖剪刀(世界精密仪器公司,目录号:14192)
  3. 学生Vannas剪刀(世界精密仪器公司,目录号:501777)
  4. 精密Vannas剪刀(世界精密仪器公司,目录号:500086)
  5. 弯曲的细Vannas剪刀(世界精密仪器,目录号:501232)
  6. 2x Dumont No.5镊子(World Precision Instruments,目录号:500085)
  7. Zeiss立体显微镜Stemi2000(Zeiss,目录号:455052)
  8. 格兰特水浴(科学实验室用品,目录号:BAT3310)
  9. Stuart SSL4跷跷板(科学实验室用品,目录号:MIX2066)
  10. 细胞培养培养箱(实验室专用)
  11. PIPETMAN Classic P10(Gilson,目录号:F144802)
  12. PIPETMAN Classic P100(Gilson,目录号:F144802)
  13. PIPETMAN Classic P200(Gilson,货号:F144802)
  14. PIPETMAN Classic P1000(Gilson,货号:F144802)
  15. 锥形管离心机5810 R摆动铲斗(Eppendorf,型号:5810 R)
  16. 玻璃钻石切割工具(Amazon)
  17. 文化罩

程序

该过程如图1所示。
注意:用于解剖和隔离SAN的储备溶液可以在隔离的前一天(配方1、2、4、5和7)准备,因为大约需要1个小时才能完成。溶液应保存在冰箱中(约4-8°C),并可以隔离长达一周。对于工作溶液,例如酶溶液,建议当天进行。使用前(〜15-20分钟),所有工作溶液都需要在37°C下预热(在水浴中)。至关重要的是,将CaCl 2 (10 µl 100 mM储备液至2.5 ml最终浓度400 µM)添加到酶溶液中,否则组织的消化会受到损害。


图1.从小鼠心脏分离SAN细胞的协议示意图


  1. 制备
    1. 解剖SAN之前,请准备以下派热克斯玻璃管:
      1. 2 x 2.5 ml低Ca 2 + / Mg 2 + 溶液(配方2);
      2. 1 x 2.5 ml酶溶液(上面的食谱3和注释);
      3. 4 x 2.5 ml的KB溶液(配方4)。
    2. 装有预热的Tyrode解决方案的烧杯(配方1)。
    3. 2个带有预热Tyrode溶液的解剖皿。
    4. 肝素/甲苯噻嗪/氯胺酮注射液(方案8)。
    5. 组装解剖所需的工具(解剖剪刀,细镊子,Vannas剪刀)。
    6. 准备一个带有层粘连蛋白涂层盖玻片的12孔板。用每张盖玻片涂上1 µl层粘连蛋白涂层盖玻片,并使用P200尖端铺开,并在盖掉盖子的情况下在培养罩中干燥。

  2. 从胸腔摘除心脏
    1. 腹腔注射赛拉嗪/氯胺酮/肝素混合物麻醉小鼠(遵守研究所指南)。
    2. 将鼠标仰卧(图2),并用70%乙醇喷洒胸部区域。使用解剖剪刀切开肋骨笼,打开胸腔,用镊子将其固定在主动脉弓上,然后用学生的Vannas弹簧剪刀将其切在镊子上方,以切除完整的心脏。不必担心会取出心脏外组织,因为以后可以清洗。在装满Tyrode溶液的烧杯中冲洗心脏。注意不要割伤心脏的心耳区域。
    3. 将心脏,用昆虫针固定在顶点的心放在浸有Tyrode溶液的解剖皿上。应当将心脏放置在鼠标胸腔中(如图2所示)。在显微镜下去除非心脏组织,例如肺,胸腺,结缔组织和脂肪。使用Vannas剪刀在心室上做一个小切口,并用装满Tyrode溶液的Pasteur移液管冲洗心脏。确保避免气泡。
    注意:如果小心不要压伤心脏组织,则可以在不麻醉的情况下通过颈椎脱位术对小鼠实施安乐死。肝素将防止血液凝结并使结节的解剖/清洁更加容易。用来冲洗心脏的Tyrode溶液可以在RT或预热的情况下进行-我们发现这两种情况之间隔离的组织质量没有差异。


    图2.解剖小鼠心脏并分离SAN。 A.将麻醉过的小鼠置于仰卧位置,并在指示的位置(虚线)进行切口以进入the腔。B.取下肋骨显示心脏的位置。通过切开主动脉弓来解剖心脏。切除前或切除后可以取出肺。C.切除后的心脏显示左右心房附件(RA和LA),左右心室(RV和LV)和主动脉(Ao)。D.心脏固定在解剖盘上-虚线显示了在不损伤SAN的情况下将心房/淋巴结与心室分开的位置。E.从心室分离后,完整的SAN,RA和LA。在将上腔室与心室分离之前,使用在左心室上看到的切口从心脏冲洗血液。F和G.去除脂肪和结缔组织后的SAN(虚线区域内)和周围组织。CT,终末cr。IAS,房间隔。

  3. SAN解剖
    1. 在显微镜下,用学生Vannas剪刀小心地在心室和心房之间的交界处做一个水平切口(见图2)。心室应停止跳动并可以丢弃。带有心房附件和传导组织的上腔室应继续搏动。
    2. 翻转心房/传导组织,使主动脉面向您,左侧的右侧心房。
    3. 使用昆虫针将心房/传导组织小心地钉在干净的含有Tyrode溶液的Sylgard解剖皿(配方9)中(见图2)。有目的地在左,右心房,主动脉边缘(参见图2)或上腔静脉和下腔静脉(如果有)的边缘放置针脚。不要过度拉伸组织。如有必要,请使用细的Vannas剪刀小心取出剩余的心室组织。
    4. 一旦组织没有心室肌,切开右心耳袋并重新固定以显示Cristae terminalis(CT)。在房间隔和the末端之间将清晰可见SAN(透明)(见图2)。
    5. 小心地从SAN下方移除所有脂肪/结缔组织。
    6. 最后,用Vannas细剪刀小心地切下SAN,然后转移到装有预热的低Ca 2 + / Mg 2 + 溶液的Pyrex试管中。
    注意:上腔静脉和下腔静脉并非总是在每次准备中都可见,但是主动脉应该可见,并且非常适合固定在顶部。可使用结缔组织或左右心耳的一部分轻松固定底端。清洁组织时,请使用锋利的镊子(不带钩子)和弯曲的Vannas剪刀,这些剪刀可让您从组织上切除并防止损坏结节。在清洁淋巴结的下面时要特别小心。要尽可能彻底地执行此操作,可能需要松开底端(或顶端)并翻转以方便操作。该节点通常会持续跳动一段较长的时间(约10-20分钟),这在解剖前几次尝试时非常重要,因为它可以可视化感兴趣的区域。如果节点停止跳动,可以通过在其上轻轻吸取新鲜的Tyrode溶液来重新启动它。

  4. 酶消化SAN组织(请参见示意图1)
    所有溶液应在37°C的水浴中预热。
    1. 将SAN组织放入预先准备的含低Ca 2 + / Mg 2 + 溶液的Pyrex管中。在37°C(在水浴中)孵育2分钟。使用经过火抛光的巴斯德移液器小心地将组织转移到第二个低Ca 2 + / Mg 2 + 管中,持续4分钟。
    2. 在37°C下将SAN组织转移到酶溶液中10-15分钟。每隔2-3分钟用抛光的巴斯德吸管手动搅拌。组织应开始看起来在结构上较不僵硬。
    3. 在KB溶液中将组织洗净4次。前3次洗涤应持续2分钟,然后进行第四次和最后一次洗涤4分钟。
    4. 将组织和KB溶液从最终清洗液转移到5或10 ml玻璃小瓶中。用适当大小(请参见注释)的火抛光玻璃巴斯德移液器轻轻擦拭(上下吸移)组织约30-60 s,避免产生气泡。让细胞悬液在室温下静置5分钟。

  5. 重新适应SAN单元
    1. 将磨碎的细胞悬液转移到15 ml Falcon管中。
    2. 将以下体积的NaCl / CaCl 2 溶液(配方5)累计添加到装有细胞悬液的Falcon管中,并置于摇动平台上。
      25 µl,4分钟
      50 µl,持续4分钟
      70 µl,4分钟
      90微升4分钟
    3. 将360 µl Tyrode-BSA(配方6)添加到细胞悬液中,并放在摇杆上4分钟。
    4. 将细胞悬液在〜60-70 x g 下离心7分钟。应该有一小片细胞团。
    5. 使用1毫升Gilson Pipetman小心除去2-2.2毫升上清液(应保留约0.8-1毫升上清液)。
    6. 用1毫升Gilson Pipetman将细胞沉淀轻轻重悬在剩余的上清液中
      并在预先制备的层粘连蛋白包被的盖玻片(12孔培养板内)中大约等量(〜60-80 µl)放置。将板在37°C(95%O 2 / 5%CO 2 )细胞培养培养箱中孵育约30-45分钟,以使细胞粘附到盖玻片上。
    7. 在显微镜下检查细胞(使用20倍物镜),以查看细胞是否可行-细胞应如此。添加1 ml /孔的预热存储溶液(配方7)或Tyrode-BSA。现在可以使用细胞了。
      从形态上讲,SAN细胞由3种类型组成:纺锤形,蜘蛛形和细长形(请参见图3)。
      注意:我们发现将细胞放在Tyrode-BSA中与将它们放入存储溶液中一样有效。总细胞数可以根据制剂的成功而变化,但是平均而言,我们希望每种制剂都能看到约100-120(每片盖玻片5-10)个活细胞。


      图3.小鼠心脏中三种类型的急性分离的单个SAN细胞的形态。 A.纺锤形。B.蜘蛛状。这些单元具有1个或多个进程。C.细长的纺锤形。健康细胞会自发搏动。比例尺:20 µm。

菜谱

  1. 蒂罗德溶液
    140 mM 氯化钠
    5.4 mM KCI
    1.2 mM KH 2 PO 4
    1.8 mM CaCl 2
    1.2 mM 氯化镁 2
    5 mM HEPES-NaOH
    5.5 mM D葡萄糖
    如果pH为碱性,则用NaOH或HCl将pH调节至7.4
  2. 低'Ca 2 + / Mg 2 + '解决方案
    140 mM氯化钠
    5.4 mM KCI
    1.2 mM KH 2 PO 4
    0.2 mM CaCl 2
    0.5 mM 氯化镁 2
    50 mM 牛磺酸
    5 mM HEPES-NaOH
    5.5 mM D葡萄糖
    1 mg / ml BSA
    如果pH为碱性,则用NaOH或HCl将pH调节至6.9
  3. 酶溶液(由2.5 ml低Ca 2 + / Mg 2 + 溶液制成[配方2])
    1 mg / ml II型胶原酶
    1 mg / ml 蛋白酶XIV
    0.5 mg / ml 弹性蛋白酶
    10μl的100 mM CaCl 2 (终浓度400μM)
  4. Kraftsbuhe'KB'培养基
    70 mM L-谷氨酸
    20 mM KCI
    80 mM KOH
    10 mM KH 2 PO 4
    10 mM牛磺酸
    10毫米HEPES-KOH
    1 mg / ml BSA
    用KOH调节pH值至7.4
  5. 重新适应解决方案
    10 mM 氯化钠
    1.8 mM CaCl 2  
  6. Tyrode-BSA
    提洛德溶液(配方1)
    1毫克/毫升BSA
  7. 存储解决方案
    100 mM 氯化钠
    35 mM KCI
    4 mM KH 2 PO 4
    1.3 mM CaCl 2
    0.7 mM氯化镁 2
    2毫米牛磺酸
    14 mM 谷氨酸钾
    0.2 mM (±)D-β-丁酸
    1 mg / ml BSA
    用NaOH调节pH至7.4
  8. 肝素/氯胺酮/甲苯噻嗪注射液
    1. 肝素
      在无菌盐水中将肝素储备液(例如,1,000 IU / ml)稀释至200 IU / ml,在4 ml盐水中稀释1 ml肝素。此解决方案可以在冰箱中保存长达4周。使用〜1 IU / g(鼠标重量)。例如,对于20 g小鼠,每100 ul 200 IU / ml肝素
    2. 氯胺酮
      使用0.5毫克/克。例如,对于20克小鼠,每100毫升100毫克/毫升的储备
    3. 赛拉嗪
      使用0.1毫克/克。例如,对于20 g小鼠,100 µl 20 mg / ml的储备液
    4. 进行注射时,每只小鼠的肝素/氯胺酮/甲苯噻嗪混合物为300 µl至500 µl
  9. 希尔加德解剖盘
    1. 根据制造商的说明,使用Sylgard 170-硅橡胶(Dow Corning)制作解剖盘
    2. 将部分A和B在培养皿(100 x 21 mm)中等量混合。用量取决于所需的厚度(对于50%的深度,我们建议足够)
    3. 将Sylgard混合物在37°C的温度下放置至少24 h(可以在室温下完成,但设置时间更长)

致谢

我们要感谢BHF的资助(RG / 15/15/311742)。这项工作得到了位于Barts的NIHR生物医学研究中心的协助。特别感谢Mangoni博士让Nobles博士访问他的实验室以学习鼠SAN心肌细胞分离的程序。该协议是根据Mangoni和Nargeot(2001)的协议改编/修改的。

利益争夺

我们没有利益冲突。

伦理

所有实验均按照英国内政部法规(由项目许可证PE9055EAD覆盖)和美国国立卫生研究院(NIH第85-23号出版物,1996年修订)发布的《实验动物的护理和使用指南》进行。 )。

参考文献

  1. DiFrancesco,D.,Ferroni,A.,Mazzanti,M。和Tromba,C。(1986)。从兔窦房结分离的细胞中的超极化激活电流(if)的属性。 J生理学 377:61-88。
  2. Mangoni,ME和Nargeot,J.(2001)。分离的小鼠窦房细胞中超极化激活电流(I(f))的属性。 Cardiovasc Res 52(1):51-64。
  3. Noble D.和Tsien,RW(1968)。心脏浦肯野纤维中慢钾电流的动力学和整流特性。 J生理学 195(1):185-214。
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Copyright: © 2020 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Aziz, Q., Nobles, M. and Tinker, A. (2020). Acute Isolation of Cells from Murine Sino-atrial Node. Bio-protocol 10(1): e3477. DOI: 10.21769/BioProtoc.3477.
  2. Aziz, Q., Finlay, M., Montaigne, D., Ojake, L., Li, Y., Anderson, N., Ludwig, A. and Tinker, A. (2018). ATP-sensitive potassium channels in the sinoatrial node contribute to heart rate control and adaptation to hypoxia. J Biol Chem 293(23): 8912-8921.
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