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Dec 2020
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Protocol for Isolation of Cardiomyocyte from Adult Mouse and Rat
分离成年小鼠和大鼠的心肌细胞   

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Abstract

The isolation of intact single adult cardiomyocytes from model animals, mouse and rat, is an essential tool for cardiac molecular and cellular research. While several methods are reported for adult mouse cardiomyocyte isolation, the viability and yield of the isolated cells have been variable. Here, we describe step-by-step protocols for high viability and yield cardiomyocyte isolation from mouse and rat, based on the use of a stable pressure Langendorff perfusion system. After the animal is euthanized or terminally anesthetized, the heart is removed from the chest and subject to Langendorff perfusion. Then, the heart is digested by perfusion with collagenase and hyaluronidase. After thorough digestion, the cardiomyocytes are dispersed and gradually recovered, the extracellular Ca2+ concentration adjusted, and cells are then ready for use. This protocol will facilitate research that requires isolated adult mouse and rat cardiomyocytes.

Keywords: Heart (), Adult Cardiomyocyte (成年鼠心肌细胞), Langendorff Perfusion (朗根多夫灌注), Mouse (小鼠), Rat (大鼠)

Background

Cardiomyocytes isolated from adult mice and rats are a key tool for studying cardiac physiology and pathology, as well as their pharmacology and toxicology. Isolation of high-quality cardiomyocytes is the most important factor for successful experiments of this kind. The general protocol for adult rodent cardiomyocyte isolation has been well summarized by Louch et al. (2011). The rat cardiomyocyte has been isolated for almost a half-century (Powell and Twist, 1976). While the mouse cardiomyocyte is more sensitive to enzyme digestion, it has greater fragility during the isolation procedure, and its use has become practical only in subsequent decades (Zhou et al., 2000). More recently, several mouse cardiomyocytes isolation method papers have been published with modified buffers, enzymes, etc. (Li et al., 2014; Roth et al., 2014; Judd et al., 2016).


To date, all the published cardiomyocyte isolation methods are based on Langendorff retrograde perfusion through the aorta with enzyme solutions. There are two Langendorff perfusion methods that are well used: 1) in stable flow perfusion, a fine peristaltic pump is used to pump the solutions to the Langendorff perfused heart; 2) in stable pressure perfusion, gravity is used to drive the buffers into the perfused heart. Because of differences in the use of the perfusion system, digestion enzymes, perfusion buffer, etc., there is no one universal method to produce a large yield of high quality, viable cardiomyocytes from adult rodent hearts. In this protocol, based on our experience with both stable flow perfusion (Zhang et al., 2013) and stable pressure perfusion (Zhang et al., 2017, 2018, 2020), we recommend using the stable pressure Langendorff perfusion system, and we present herein optimized adult mouse and rat cardiomyocyte isolation protocols that are easy to follow and result in high yield and cell viability.

Materials and Reagents

  1. 23 G needle (BD, catalog number: 305194), trimmed the tip and covered by a thin silastic tube (Warner, catalog number: PE-50)

  2. 18 G needle (BD, catalog number: 05196), trimmed the tip and covered by a thin silastic tube (Warner, catalog number: PE-160)

  3. 50 mL beaker (Fisher, catalog number: FB-100-50)

  4. 60 mm diameter tissue culture dish (Falcon, catalog number: 353002)

  5. 1 mL Pipette tips (Thermo Scientific, catalog number: 94056710)

  6. NaCl (Fisher, catalog number: S271-3)

  7. KCl (Fisher, catalog number: P217-500)

  8. HEPES (Fisher, catalog number: BP310-100)

  9. NaH2PO4·H2O (Fisher, catalog number: BP330-500)

  10. KH2PO4 (J.T. Baker, catalog number: 3246-01)

  11. MgSO4·7H2O (Sigma, catalog number: M2773-500G)

  12. Glucose (Fisher, catalog number: D16-1)

  13. 2,3-Butanedione monoxime, BDM (Alfa Aesar, catalog number: A14339)

  14. Taurine (ACROS ORGANICS, catalog number: 166541000)

  15. L-Glutathione, Reduced (UBPBio, catalog number: P1030-100)

  16. CaCl2 (Fisher, catalog number: BP510-100)

  17. Bovine serum albumin, BSA (Sigma, catalog number: A7906-100)

  18. Collagenase Type 2 (Warthington, catalog number: LS004177)

  19. Hyaluronidase from bovine testes (Sigma, catalog number: H3506-5G)

  20. Pentobarbital sodium (Oak Pharmaceuticals, catalog number: NDC 76478-501-50)

  21. Heparin (Fresenius Kabi, catalog number: 63323-540-11)

  22. Fetal bovine serum (HyClone, SH30396.03)

  23. Oxygen (Praxair, with Prostar platinum oxygen modulator)

  24. Mouse cardiomyocyte isolation buffers (see Recipes)

  25. Rat cardiomyocyte isolation buffers (see Recipes)

Equipment

  1. Forceps (World Precision Instruments, catalog number: 15915)

  2. Langendorff perfusion system with ~90 cm water pressure with an isolation rig (Double Baker Warming Coil, Harvard Apparatus, catalog number: PY8 50-8382) (Figure 1)

  3. Circulating Water Bath (Julabo, CORIO CD)

  4. Water Bath (Precision, catalog number: 51221048)

  5. Pump for circulating the enzyme buffer (Fisher, Minipump Variable Flow, catalog number: EF20714B)

  6. Centrifuge for 15 ml tube (Thermo, CENTRA CL2)

Procedure

  1. The mouse and rat cardiomyocyte isolation protocols share the same experimental device settings (Figure 1, and Video 1):

    1. Langendorff perfusion system with ~90 cm water pressure (Figure 1A, note height of the water column). Using the Double Baker Warming Coil to switch between the perfusion buffer and enzyme digestion buffer (Figure 1B).

    2. Clean the isolation rig (Figure 1, indicated with red box) and tubes with 70% ethanol one time by filling the left 50 mL tube to the full level with 70% ethanol, after which the ethanol will go to all the left tubes and the left part of the rig. Repeat for the right tube. Drain the 70% ethanol. Fill the tubes with autoclaved H2O and drain two times.

    3. Turn on the circulating water bath (Figure 1A, lower left) and the regular 37°C water bath (see Video 1).

    4. The peristaltic pump is used only to circulate perfusion enzyme buffer released from the heart back into the 50 mL perfusion tube, as the former fills and the latter is exhausted.



    Figure 1. Adult cardiomyocyte isolation system.

    (A) The heart perfusion setup. (B) A zoomed view of the isolation rig and its connections. The needle with the cannulated heart will be connected to the bottom part of the rig (see the arrow indicated by “To Needle with Cannulated Heart”).


    Video 1. Perfusion system introductionPart I: Adult mouse cardiomyocyte isolation


  2. Part I: Adult mouse cardiomyocyte isolation

    1. Prepare the isolation and digestion buffers (see Recipes for Mouse cardiomyocyte isolation buffers). Fill the system with 80 mL of mouse cardiomyocyte isolation buffer into the left tube (Figure 1A, the total volume of 50 mL syringe tube plus the tubes system is approximately 110 mL) and 50 mL of enzyme digestion buffer into the right tube (Figure 1A) and bubble with oxygen delivered by tubing that reaches the bottom of both the left and the right 50 mL tubes (Figure 1A). Connect a 23 G needle covered by a thin plastic tube (to prevent a loose tie, Figure 2A) to a syringe filled with perfusion buffer.



      Figure 2. Mouse heart needle cannulation and perfusion.

      (A) A 23 G needle with a silastic tube cover to facilitate the seal to the aorta and prevent a loose tie. (B) A typical perfused mouse heart. The heart is cannulated to the 23 G needle and ligated using 6-0 silk thread.


    2. Euthanize the mouse by cervical dislocation. Spray the mouse chest with 70% ethanol.

    3. Open the chest cavity and quickly take out the heart with approximately 2 mm of aorta. Put the heart in the mouse cardiomyocyte isolation buffer on ice. Gently press the heart to remove blood in the chambers.

    4. Cannulate the aorta to the 23 G tube-covered needle and tie the aorta to the cannula with 6-0 silk thread. The tip of the cannula should be just above the aortic valve. Gently flush the heart with 2–3 mL perfusion buffer to ensure that there is no leak and to flush out most of the blood (see Video 2).

      Note: The total time to cannulate the heart should be less than 1 min.


      Video 2. Mouse heart canulation


    5. Transfer the cannulated heart to the Langendorff perfusion system, attaching the 23 G needle (and heart) to the fitting at the lower end of the rig (Figures 1B and 2B). Perfuse the heart with mouse cardiomyocyte isolation buffer from the left tube for approximately 5 min, during which time approximately 15 mL of isolation buffer should flow through. During this time, gently press the heart to further remove any blood left in the chamber until the efflux is clear.

      Note: Any blood left in the heart can neutralize the digestion buffer enzymes and lower the digestion efficiency.

    6. Switch to the digestion buffer (right tube, 250 U/mL collagenase II + 0.2 mg/mL hyaluronidase) by turning the Buffer Switch Knob (Figure 1B) 180°, catch the efflux digestion buffer in a 60 mm tissue culture dish; turn on the circulating pump (Figure 1A) to gradually add this digestion buffer back into the right tube containing fresh digestion buffer (Figure 1A). At that point, also add 40 µL of CaCl2 (100 mM stock) to the circulating digestion buffer, to result in ~80 µM CaCl2. Thus, the heart will be digested by buffer without Ca2+ at the very beginning, but as the digestion buffer with Ca2+ gradually reaches the heart, the heart accommodates to the high efficiency 80 µM CaCl2 digestion buffer.

      Note: 80 µM Ca2+ accelerates the digestion but does not damage the cells.

    7. Perfuse with digestion buffer for 20–30 min until the heart becomes soft.

      Note: The heart should swell and look soft, pinkish, but not pale white, as the latter means the perfusion buffer does not reach the heart tissue. A pale white heart could be an issue with the aorta cannulation or blood clotting in the coronary arteries. The constant-pressure flow rate will increase during perfusion as the heart swells.

    8. Excessive digestion will reduce the cardiomyocyte viability. The endpoint of digestion should therefore be judged by the softness of the heart. When very soft and flexible, detach the heart from the cannulated syringe needle, remove the atria, and tear apart the ventricle tissue into 3–4 pieces in 5 mL digestion buffer (from the circulated perfused digestion buffer) using forceps in the collection dish.

    9. Leave the tissue in 5 mL of digestion buffer for 5 min. Pipette the tissue up and down 5–6 times in a 1 mL pipette tip.

      Note: Cut the end of the 1 mL tip to approximately 3 mm diameter to make sure that the tissue fragments easily go through the tip).

      The cells will disperse from the tissue into the digestion buffer: the cells will be in the buffer, and tissue clumps will be in the bottom of the dish. Collect the suspended isolated cells with a 1 mL pipette tip to a 15 mL tube, and then add 2 mL of stopping buffer to the cells in the tube.

    10. Add 5 mL of enzyme digestion buffer to the leftover tissue to further digest 5 min more and disperse the cells as in step 9. Collect the dispersed cells and add 2 mL of stopping buffer. Repeat steps 9 and 10 until the tissue becomes white and only connective tissue is left.

    11. Combined all the isolated cells. Centrifuge at 100 × g for 20 s or let the cells settle down for 10 min.

    12. Remove the supernatant, add 10 mL of fresh stop buffer and resuspend the cells.

    13. Add 10 µL of CaCl2 (100 mM) four times (40 µL total) with a 5 min interval between each addition; each time, mix the solution by gently pipetting up and down three times. This accomplishes gradual Ca2+ restoration to avoid high Ca2+ concentration stress.

    14. Centrifuge the cells at 100 × g for 20 s or let the cells settle down for 10 min.

    15. Remove the supernatant and resuspend the cells in the appropriate buffer or medium for your experimental use. See the primary isolated mouse cardiomyocytes’ appearance, Figure 4A.


  1. Part II: Adult rat cardiomyocyte isolation

    The procedure for rat cardiomyocyte isolation is very similar to that of mouse cardiomyocyte isolation. The major differences are in the perfusion buffer recipe, enzyme amount, and cutting of the ventricle tissue into small chunks.

    1. Prepare the isolation and digestion buffers (see Recipes for rat cardiomyocyte isolation buffers). Fill the system with 80 mL of 1 mM Ca2+ perfusion buffer (left tube, Figure 1A) and 50 mL of Ca2+ free perfusion buffer (right tube, Figure 1A) and bubble with oxygen by O2 tubes reaching the bottom of both left and right 50 mL tubes (Figure 1A). Connect an 18 G needle covered by a thin silastic tube (to prevent a loose tie around the aorta, Figure 3) to a syringe filled with 5 mL of 1 mM Ca2+ perfusion buffer.



      Figure 3. Rat heart cannulation needle.

      An 18 G needle covered with silastic tubing to prevent a loose tie.


    2. Anesthetize the rat by i.p. injection with pentobarbital sodium (150 mg/Kg) and heparin (10,000 U in 0.5 mL) to prevent blood clotting. Pinch the foot pad. When the rat has no response, then move to the next step.

    3. Spray the rat chest with 70% ethanol. Open the chest wall, cut the ribs and quickly remove the heart with 3–5 mm aorta attached. Put the heart in 1 mM Ca2+ perfusion buffer on ice. Gently press the heart to remove blood in the chambers.

    4. Cannulate the aorta with the 18 G needle and tie the aorta to the cannula with 4-0 silk thread. The tip of the cannula should be just above the aortic valve. Flush 3–4 mL buffer into aorta to check that there is no leak and to remove most of the blood (see Video 3). Transfer the heart to the isolation rig.


      Video 3. Rat Heart Canulation


    5. Perfuse with 1 mM Ca2+ isolation buffer for 5 min to remove any blood left in the heart chamber, until the efflux is clear.

    6. Switch to Ca2+-free perfusion buffer for 5 min. The heart will gradually stop beating.

    7. Add 20 mL of concentrated digestion buffer (~6,500 U collagenase II and 12 mg hyaluronidase) to the Ca2+-free perfusion buffer that remains in the tube (~60 mL). The total volume is 80 mL.

    8. Catch the efflux digestion buffer with a 50 mL beaker and submerge the heart in the digestion buffer. Recirculate the digestion buffer, as in the mouse protocol.

    9. After 5 min, add 150 µL of 100 mM CaCl2 three times at 1 min intervals into the digestion buffer to gradually increase the Ca2+ concentration. The final Ca2+ concentration will be approximately 500 µM to accelerate the digestion.

    10. The heart should swell and look soft, pinkish, but not pale white, as the latter means that the perfusion buffer has not reached the heart tissue. A pale white heart could be an issue with the aorta cannulation or blood clotting in the coronary arteries. The flow rate will increase during perfusion as the heart swells. Total digestion time is 40–60 min.

    11. When the heart becomes soft, detach the heart from the cannulated syringe needle, remove the atria and aorta, and cut the ventricle tissue into small chunks (~2 mm cubes).

    12. Add 15 mL of enzyme digestion buffer (from the circulated digestion buffer) and gently swirl in a 37°C water bath for 5 min.

    13. After the first swirl, do not pipette the chunks, but gently pipette the first swirl solution, which contains mostly dead cells, and discard it. Add 15 mL of enzyme digestion buffer, swirl the 2nd time for 5 min, and now pipette the chunks up and down to disperse the cardiomyocytes. The cells will be suspended in the buffer, and the tissue will be at the bottom of the dish. Collect the dispensed cells with the 1 mL pipette and add to a 15 mL tube to which you then add 2 mL of stopping buffer. Add 15 mL of enzyme digestion buffer to the rest of the chunks for the 3rd digestion. Repeat 3–4 times till the chunks become white and only connective tissue is left. Combine all the isolated cells. Centrifuge at 100 × g for 20 s. Resuspend the cells in 20 mL of stopping buffer and place 10 mL in each of two 15 mL tubes. Add 25 µL of 100 mM CaCl2 in each tube three times at 5 min intervals to gradually restore the Ca2+. After each addition, gently turn the 15 mL tube upside down to mix the Ca2+.

    14. Centrifuge at 100 × g for 20 s. Resuspend the cells in the appropriate buffer or medium for the experimental use. See the isolated rat cardiomyocyte appearance after 24 h culture in Figure 4B.



    Figure 4. Isolated mouse and rat cardiomyocytes.

    (A) Acute isolated mouse cardiomyocytes. (B) Rat cardiomyocytes after 24 h culture.

Notes

Collagenase Type 2 activity varies between different batches. Purchase a small bottle of several batches from the vendor and test which batch works best.

Recipes

  1. Mouse cardiomyocyte isolation buffers

    1. Mouse cardiomyocyte perfusion buffer (pH 7.4, adjusted with NaOH)

      The solution contains:

      113 mM NaCl; 4.7 mM KCl; 1.2 mM MgSO4·7H2O; 0.6 mM NaH2PO4; 0.6 mM KH2PO4; 10 mM HEPES; 10 mM Glucose; 10 mM Butanedione monoxime; 5 mM Taurine

      Note: Filter all the buffers and solutions through 0.22 μm filter.

    2. Mouse heart digestion buffer

      Mouse cardiomyocyte perfusion buffer + Collagenase (300 U/mL) + Hyaluronidase (0.5 mg/mL)

    3. Mouse heart stop digestion buffer

      Mouse cardiomyocyte perfusion buffer + BSA (2.5%) + CaCl2 (0.1 mM) + Fetal Bovine Serum (5%)

    4. 100 mM CaCl2 stock solution


  2. Rat cardiomyocyte isolation buffers

    1. Rat cardiomyocyte perfusion buffer (pH 7.4, adjusted with NaOH) with or without 1 mM Ca2+

      The solution contains:

      118 mM NaCl; 4.8 mM KCl; 10 mM HEPES; 1.2 mM KH2PO4; 1.2 mM MgSO4·7H2O; 11 mM Glucose; 1.0 mM CaCl2·2H2O

      Note: For the Ca2+ free perfusion buffer, no Ca2+ added.

    2. Rat heart digestion buffer

      In 20 mL Ca2+ free perfusion buffer, add 6,500 U collagenase II and 12 mg hyaluronidase.

    3. Rat heart stop digestion buffer

      2% BSA in 0.1 mM Ca2+ perfusion buffer.

    4. 100 mM CaCl2 stock solution

Acknowledgments

This work was supported by NIA (P01AG001751) to P.S.R. and AHA (19CDA34660311), UW CTMR pilot award under P30AR074990 and Bronson Foundation intramural grant to H.Z.

These protocols are developed based on the experience gained in the laboratories of Dr. Heping Cheng and Dr. Wang Wang, to whom H.Z. is very grateful.

This protocol is linked to the Bio-protocol partner journal eLife with the original research paper DOI: 10.7554/eLife.60827, PMID: 33319746.

Competing interests

The authors declare no conflict of interest.

References

  1. Judd, J., Lovas, J. and Huang, G. N. (2016). Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes. J Vis Exp(114): 54012.
  2. Li, D., Wu, J., Bai, Y., Zhao, X. and Liu, L. (2014). Isolation and culture of adult mouse cardiomyocytes for cell signaling and in vitro cardiac hypertrophy. J Vis Exp(87): 51357.
  3. Louch, W. E., Sheehan, K. A. and Wolska, B. M. (2011). Methods in cardiomyocyte isolation, culture, and gene transfer. J Mol Cell Cardiol 51(3): 288-298.
  4. Powell, T. and Twist, V. W. (1976). A rapid technique for the isolation and purification of adult cardiac muscle cells having respiratory control and a tolerance to calcium. Biochem Biophys Res Commun 72(1): 327-333.
  5. Roth, G. M., Bader, D. M. and Pfaltzgraff, E. R. (2014). Isolation and physiological analysis of mouse cardiomyocytes. J Vis Exp(91): e51109.
  6. Zhang, H., Alder, N. N., Wang, W., Szeto, H., Marcinek, D. J. and Rabinovitch, P. S. (2020). Reduction of elevated proton leak rejuvenates mitochondria in the aged cardiomyocyte. Elife 9: e60827.
  7. Zhang, H., Gong, G., Wang, P., Zhang, Z., Kolwicz, S. C., Rabinovitch, P. S., Tian, R. and Wang, W. (2018). Heart specific knockout of Ndufs4 ameliorates ischemia reperfusion injury. J Mol Cell Cardiol 123: 38-45.
  8. Zhang, H., Shang, W., Zhang, X., Gu, J., Wang, X., Zheng, M., Wang, Y., Zhou, Z., Cao, J. M., Ji, G., Zhang, R. and Cheng, H. (2013). Beta-adrenergic-stimulated L-type channel Ca2+ entry mediates hypoxic Ca2+ overload in intact heart. J Mol Cell Cardiol 65: 51-58.
  9. Zhang, H., Wang, P., Bisetto, S., Yoon, Y., Chen, Q., Sheu, S. S. and Wang, W. (2017). A novel fission-independent role of dynamin-related protein 1 in cardiac mitochondrial respiration. Cardiovasc Res 113(2): 160-170.
  10. Zhou, Y. Y., Wang, S. Q., Zhu, W. Z., Chruscinski, A., Kobilka, B. K., Ziman, B., Wang, S., Lakatta, E. G., Cheng, H. and Xiao, R. P. (2000). Culture and adenoviral infection of adult mouse cardiac myocytes: methods for cellular genetic physiology. Am J Physiol Heart Circ Physiol 279(1): H429-436.

简介

[摘要] 从模型动物、小鼠和大鼠中分离完整的单个成年心肌细胞是心脏分子和细胞研究的重要工具。虽然报告了几种用于成年小鼠心肌细胞分离的方法,但分离细胞的活力和产量是可变的。在这里,我们描述了基于使用稳定压力Langendorff灌注系统的高生存力和产量心肌细胞从小鼠和大鼠分离的分步协议。在动物被安乐死或最终麻醉后,将心脏从胸部取出并接受Langendorff灌注。然后,通过用胶原酶和透明质酸酶灌注来消化心脏。彻底消化后,心肌细胞分散并逐渐恢复,调整细胞外Ca 2+浓度,即可使用。该协议将促进需要分离的成年小鼠和大鼠心肌细胞的研究。


[背景] 从成年小鼠和大鼠中分离出的心肌细胞是研究心脏生理学和病理学及其药理学和毒理学的关键工具。高质量心肌细胞的分离是此类实验成功的最重要因素。 Louch等人已经很好地总结了成年啮齿动物心肌细胞分离的一般方案。 (2011 年) 。大鼠心肌细胞已被分离了近半个世纪(Powell 和 Twist,1976) 。虽然小鼠心肌细胞对酶消化更敏感,但在分离过程中具有更大的脆弱性,并且它的使用直到随后的几十年才变得实用(Zhou 等人, 2000) 。最近,已经发表了几篇小鼠心肌细胞分离方法论文,其中包括改良的缓冲液、酶等。 (李等人, 2014;罗斯 等人, 2014;贾德等人, 2016) 。
迄今为止,所有已发表的心肌细胞分离方法都是基于Langendorff逆行灌注通过主动脉用酶溶液。有两种很好使用的Langendorff灌注方法:1)在稳定流动灌注中,使用精细蠕动泵将溶液泵送到Langendorff灌注心脏; 2) 在稳定的压力灌注中,重力被用来驱动缓冲液进入灌注的心脏。由于灌注系统、消化酶、灌注缓冲液等的使用不同,没有一种通用的方法可以从成年啮齿动物心脏中大量生产高质量、有活力的心肌细胞。在本协议中,根据我们在稳定流量灌注(Zhang et al ., 2013)和稳定压力灌注(Zhang et al ., 2017, 2018, 2020)方面的经验,我们推荐使用稳定压力Langendorff灌注系统,我们本文提出了优化的成年小鼠和大鼠心肌细胞分离方案,这些方案易于遵循并导致高产量和细胞活力。

关键字:心, 成年鼠心肌细胞, 朗根多夫灌注, 小鼠, 大鼠

材料和试剂
1. 23 G针(BD,目录号:305194),修剪尖端并用薄硅橡胶管覆盖(Warner,目录号:PE-50)
2. 18 G针(BD,目录号:05196),修剪尖端并用薄硅橡胶管覆盖(Warner,目录号:PE-160)
3. 50 mL烧杯(Fisher,目录号:FB-100-50)
4. 60 mm直径的组织培养皿(Falcon,目录号:353002)
5. 1 mL移液器吸头(Thermo Scientific,目录号:94056710 )
6. NaCl(Fisher,目录号:S271-3)
7. KCl (Fisher,目录号:P217-500)
8. HEPES ( Fisher,目录号:BP310-100)
9. NaH 2 PO 4 · H 2 O(Fisher,目录号:BP330-500)
10. KH 2 PO 4 (JT Baker,目录号:3246-01)
11. MgSO 4 · 7H 2 O(Sigma,目录号:M2773-500G)
12. 葡萄糖(Fisher,目录号:D16-1)
13. 2,3-丁二酮一肟,BDM(Alfa Aesar ,目录号:A14339)
14. 牛磺酸(ACROS ORGANICS,目录号:166541000 )
15. L-谷胱甘肽,还原( UBPBio ,目录号:P1030-100 )
16. CaCl 2 (Fisher,目录号:BP510-100)
17. 牛血清白蛋白,BSA(Sigma,目录号:A7906-100)
18. 2型胶原酶( Warthington ,目录号:LS004177)
19. 来自牛睾丸的透明质酸酶(Sigma,目录号:H3506-5G)
20. 戊巴比妥钠(Oak Pharmaceuticals,目录号:NDC 76478-501-50)
21. 肝素(Fresenius Kabi ,目录号:63323-540-11)
22. 胎牛血清( HyClone ,SH30396.03)
23. 氧气(普莱克斯,带有 Prostar 铂氧调节器)
24. 小鼠心肌细胞分离缓冲液 (见食谱)
25. 心肌细胞分离缓冲液中的R (见食谱)


设备


1. 镊子(World Precision Instruments,目录号:15915 )
2. 具有~90 cm水压的Langendorff灌注系统和隔离装置(Double Baker Warming Coil,Harvard Apparatus,目录号: PY8 50-8382)(图1)
3. 循环水浴 ( Julabo , CORIO CD)
4. 水浴(Precisi on,目录号: 51221048)
5. 用于循环酶缓冲液的泵(Fisher,Minipump Variable Flow,目录号: EF20714B)
6. 用于 15 ml 管的离心机(Thermo,CENTRA CL2)


程序


A. 小鼠和大鼠心肌细胞分离协议共享相同的实验设备设置(图 1 和视频 1):
1. Langendorff灌注系统,水压约为 90 厘米(图 1A,注意水柱的高度)。使用 Double Baker 加热线圈在灌注缓冲液和酶消化缓冲液之间切换(图 1B)。
2. 用 70% 乙醇将左侧 50 mL 管填充到满水平,用 70% 乙醇清洁隔离装置(图 1,用红色框表示)和管一次,之后乙醇将进入所有左侧管和钻机的左侧部分。重复右管。排出 70% 乙醇。用高压灭菌的 H 2 O填充管并排放两次。
3. 打开循环水浴(图 1A,左下)和常规 37 °C水浴(参见视频 1)。
4. 蠕动泵仅用于将从心脏释放的灌注酶缓冲液循环回 50 mL 灌注管,因为前者充满,后者耗尽。




 
图 1.成人心肌细胞分离系统。
(A) 心脏灌注设置。 (B) 隔离装置及其连接的放大视图。带有空心心脏的针头将连接到钻机的底部(参见“带空心心脏的针头”指示的箭头)。


 
视频 1. 灌注系统介绍




B. 第一部分:成年小鼠心肌细胞分离
1. 准备分离和消化缓冲液(参见小鼠心肌细胞分离缓冲液配方)。将 80 mL 的小鼠心肌细胞分离缓冲液填充到左管中(图 1A,50 mL 注射器管加上管系统的总体积约为 110 mL)和 50 mL 的酶消化缓冲液进入右管(图 1A ) 和通过管道输送的氧气气泡,到达左侧和右侧 50 mL 管的底部(图 1A)。将由细塑料管覆盖的 23 G 针头(以防止松散的领带,图 2A)连接到充满灌注缓冲液的注射器。
 
图 2.小鼠心脏针插管和灌注离子。 
(A) 带有硅橡胶管盖的 23 G 针头,便于密封主动脉并防止领带松动。 (B) 典型的灌注小鼠心脏。将心脏插管至 23 G 针并使用 6-0 丝线结扎。


2. 通过颈椎脱位安乐死鼠标。用 70% 乙醇喷洒鼠标胸部。
3. 打开胸腔,用大约 2 mm 的主动脉迅速取出心脏。将心脏放在冰上的小鼠心肌细胞隔离缓冲液中。轻轻按压心脏以去除腔室中的血液。
4. 将主动脉插到 23 G 管覆盖的针上,并用 6-0 丝线将主动脉绑在插管上。套管的尖端应刚好在主动脉瓣上方。用 2-3 mL 灌注缓冲液轻轻冲洗心脏,以确保没有泄漏并冲洗掉大部分血液(参见视频 2)。
注意:对心脏进行插管的总时间应少于 1 分钟。


 
视频2. 小鼠心脏插管


5. 将空心心脏转移到Langendorff灌注系统,将 23 G 针(和心脏)连接到钻机下端的配件(图 1B 和 2B)。用左管的小鼠心肌细胞隔离缓冲液灌注心脏约 5 分钟,在此期间大约 15 mL 的隔离缓冲液应流过。在此期间,轻轻按压心脏以进一步清除腔内残留的任何血液,直到流出物清除。
注意:任何留在心脏中的血液都会中和消化缓冲酶,降低消化效率。
6. 通过将缓冲开关旋钮(图 1B)旋转 180 ° ,切换到消化缓冲液(右管,250 U/mL 胶原酶 II + 0.2 mg/mL 透明质酸酶),在 60 mm 组织培养皿中捕获外排消化缓冲液;打开循环泵(图 1A),逐渐将此消化缓冲液添加回含有新鲜消化缓冲液的右管中(图 1A)。此时,还要将 40 μL 的 CaCl 2 (100 mM 库存)添加到循环消化缓冲液中,以产生 ±80 μM CaCl 2 。因此,心脏一开始会被不含 Ca 2+的缓冲液消化,但随着含有 Ca 2+的消化缓冲液逐渐到达心脏,心脏会适应高效的 80 µM CaCl 2消化缓冲液。
注意:80 µM Ca 2+可加速消化,但不会损坏细胞。
7. 用消化缓冲液灌注20-30 分钟,直到心脏变软。
注意:心脏应该肿胀并且看起来柔软、粉红色,但不是淡白色,因为后者意味着灌注缓冲液不会到达心脏组织。苍白的心脏可能是主动脉插管或冠状动脉血栓的问题。随着心脏肿胀,灌注期间恒压流速将增加。
8. 消化会降低心肌细胞的活力。 因此,消化的终点应该以心脏的柔软度来判断。当非常柔软和灵活时,将心脏与空心注射器针头分离,取出心房,然后使用收集盘中的镊子将心室组织撕成 3-4 块,放入 5 mL 消化缓冲液(来自循环灌注消化缓冲液)中。
9. 将组织留在 5 mL的消化缓冲液中 5 分钟。在 1 mL 移液器吸头中上下移取组织5-6次。
注意:将 1 mL 吸头的末端切成约 3 mm 直径,以确保组织碎片轻松穿过吸头)。
细胞将从组织分散到消化缓冲液中:细胞将在缓冲液中,组织块将在培养皿底部。用 1 mL 移液器尖端将悬浮的分离细胞收集到 15 mL 管中,然后将 2 mL 的停止缓冲液添加到管中的细胞中。
10. 将 5 mL 的酶消化缓冲液添加到剩余组织中,以进一步消化 5 分钟并分散细胞,如步骤 9 所示。收集分散的细胞并添加 2 mL 停止缓冲液。重复步骤 9 和 10,直到组织变成白色,只剩下结缔组织。
11. 结合所有分离的细胞。以 100 × g离心20 秒或让细胞静置 10 分钟。
12. 去除上清液,加入 10 mL 新鲜停止缓冲液并重新悬浮细胞。
13. 添加 10 µL 的 CaCl 2 (100 mM) 四次(总共 40 µL),每次添加之间间隔 5 分钟;每次轻轻上下吹打 3 次,混合溶液。这实现了逐渐的Ca 2+恢复以避免高Ca 2+浓度应力。
14. 以 100 × g离心细胞20 秒或让细胞静置 10 分钟。
15. 去除上清液并将细胞重新悬浮在适当的缓冲液或培养基中以供您的实验使用。参见主要分离的小鼠心肌细胞的外观,图 4A。


C. 第二部分:成年大鼠心肌细胞分离
大鼠心肌细胞分离的过程与小鼠心肌细胞分离的过程非常相似。主要区别在于灌注缓冲液配方、酶量和将心室组织切割成小块。
1. 准备分离和消化缓冲液(参见大鼠心肌细胞分离缓冲液配方)。用 80 mL 的 1 mM Ca 2+灌注缓冲液(左管,图 1A)和 50 mL 的 Ca 2+游离灌注缓冲液(右管,图 1A)填充系统,并通过 O 2管到达底部的氧气气泡左右 50 mL 管(图 1A)。将由薄硅橡胶管覆盖的 18 G 针头(以防止主动脉周围松散,图 3)连接到装有 5 mL 1 mM Ca 2+灌注缓冲液的注射器。


 
图 3.大鼠心脏插管针。 
一个 18 G 的针头,上面覆盖着硅橡胶管,以防止领带松动。


2. ip注射戊巴比妥钠 (150 mg/Kg) 和肝素 (10,000 U 在 0.5 mL)麻醉大鼠,以防止血液凝固。捏住脚垫。当大鼠没有反应时,再进行下一步。
3. 用 70% 乙醇喷洒大鼠胸部。打开胸壁,切开肋骨,快速取出附有 3-5 毫米主动脉的心脏。将心脏置于冰上1 mM Ca 2+灌注缓冲液中。轻轻按压心脏以去除腔室中的血液。
4. 用 18 G 针将主动脉插管,并用 4-0 丝线将主动脉绑在套管上。套管的尖端应刚好在主动脉瓣上方。将 3 – 4 mL 缓冲液冲洗到主动脉中,以检查是否没有泄漏并去除大部分血液(参见视频 3)。将心脏转移到隔离装置。


 
视频 3. 大鼠心脏插管


5. 用 1 mM Ca 2+隔离缓冲液灌注 5 分钟,以去除留在心室中的任何血液,直到流出物清除。
6. 切换到 Ca 2+ -free 灌注缓冲液 5 分钟。心脏会逐渐停止跳动。
7. 将 20 mL 的浓缩消化缓冲液(~6,500 U 胶原酶 II 和 12 mg 透明质酸酶)添加到留在管中的 Ca 2+无灌注缓冲液(~60 mL)中。总体积为 80 mL。
8. 用 50 mL 烧杯捕捉外排消化缓冲液,并将心脏浸入消化缓冲液中。再循环消化缓冲液,如在鼠标协议中。
9. 5 分钟后,每隔 1 分钟将 150 μL 的 100 mM CaCl 2添加到消化缓冲液中三次,以逐渐增加 Ca 2+浓度。最终的 Ca 2+浓度约为 500 µM,以加速消化。
10. 心脏应该肿胀并且看起来柔软、粉红色,但不是淡白色,因为后者意味着灌注缓冲液尚未到达心脏组织。苍白的心脏可能是主动脉插管或冠状动脉血栓的问题。随着心脏肿胀,灌注期间流速会增加。总消化时间为 40 – 60 分钟。
11. 当心脏变软时,将心脏与空心注射器针头分离,取出心房和主动脉,并将心室组织切成小块(约 2 毫米立方体)。
12. 加入 15 mL 酶消化缓冲液(来自循环的消化缓冲液)并在 37°C 水浴中轻轻旋转 5 分钟。
13. 第一次漩涡后,不要移取大块,而是轻轻移取第一次漩涡溶液,其中大部分是死细胞,然后将其丢弃。加入 15 mL 的酶消化缓冲液,第 2 次旋转5分钟,然后上下移液以分散心肌细胞。细胞将悬浮在缓冲液中,组织将位于培养皿底部。用 1 mL 移液器收集分配的细胞并添加到 15 mL 管中,然后向其中添加 2 mL 停止缓冲液。将 15 mL 的酶消化缓冲液添加到其余的块中进行第 3次消化。重复3-4次,直到大块变成白色,只剩下结缔组织。结合所有孤立的细胞。以 100 × g离心20 秒。将细胞重新悬浮在 20 mL 的停止缓冲液中,并在两个 15 mL 管中的每一个中放置 10 mL。每隔 5 分钟在每管中加入 25 μL 的 100 mM CaCl 2三次,以逐渐恢复 Ca 2+ 。每次添加后,轻轻将 15 mL 管倒置以混合 Ca 2+ 。
14. 以 100 × g离心20 秒。将细胞重新悬浮在适当的缓冲液或培养基中以供实验使用。请参阅图 4B 中 24 小时培养后分离的大鼠心肌细胞外观。


 
图 4. 分离的小鼠和大鼠心肌细胞。 
(A) 急性分离的小鼠心肌细胞。 (B) 24 小时培养后的大鼠心肌细胞。


笔记


2 型胶原酶活性在不同批次之间有所不同。从供应商处购买几批的一小瓶,并测试哪一批效果最好。


食谱


A. 小鼠心肌细胞分离缓冲液
1. 小鼠心肌细胞灌注缓冲液(pH 7.4 ,用 NaOH 调节)
解决方案包含:
113 毫米氯化钠 ; 4.7 毫米氯化钾 ; 1.2 mM MgSO 4 · 7H 2 O ; 0.6 mM NaH 2 PO 4 ; 0.6 毫米 KH 2 PO 4 ; 10 毫米 HEPES ; 10 毫米葡萄糖 ; 10 mM 丁二酮一肟; 5 毫米牛磺酸
注意:通过 0.22 μm过滤器过滤所有缓冲液和溶液。
2. 小鼠心脏消化缓冲液
小鼠心肌细胞灌注缓冲液 + 胶原酶 (300 U/mL) + 透明质酸酶 (0.5 mg/mL)
3. 小鼠心脏停止消化缓冲液
小鼠心肌细胞灌注缓冲液 + BSA (2.5%) + CaCl 2 (0.1 mM) + 胎牛血清 (5%)
4. 100 mM CaCl 2储备溶液


B. 大鼠心肌细胞分离缓冲液
1. 含或不含 1 mM Ca 2+的大鼠心肌细胞灌注缓冲液(pH 7.4,用 NaOH 调节)
解决方案包含:
118毫米氯化钠; 4.8毫米氯化钾; 10毫米HEPES ; 1.2毫米KH 2 PO 4 ; 1.2 mM MgSO 4 · 7H 2 O ; 11毫米葡萄糖; 1.0 mM CaCl 2 · 2H 2 O
注意:对于不含 Ca 2+的灌注缓冲液,不添加 Ca 2+ 。
2. 大鼠心脏消化缓冲液:
在 20 mL Ca 2+游离灌注缓冲液中,加入 6,500 U 胶原酶 II 和 12 mg 透明质酸酶。
3. 大鼠心脏停止消化缓冲液:
0.1 mM Ca 2+灌注缓冲液中的 2% BSA。
4. 100 mM CaCl 2储备溶液。


致谢


这项工作得到了 NIA (P01AG001751) 对 PSR 和 AHA (19CDA34660311)、P30AR074990 下的 UW CTMR 试点奖和布朗森基金会对 HZ 的校内赠款的支持
程和平博士和王王博士的实验室获得的经验基础上开发的,HZ 非常感谢他们。
该协议与生物协议合作伙伴期刊eLife相关联,原始研究论文 DOI:10.7554/eLife.60827,PMID:33319746。


利益争夺


作者宣称没有利益冲突。


参考


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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Zhang, H. and Rabinovitch, P. S. (2022). Protocol for Isolation of Cardiomyocyte from Adult Mouse and Rat. Bio-protocol 12(10): e4412. DOI: 10.21769/BioProtoc.4412.
  2. Zhang, H., Alder, N. N., Wang, W., Szeto, H., Marcinek, D. J. and Rabinovitch, P. S. (2020). Reduction of elevated proton leak rejuvenates mitochondria in the aged cardiomyocyte. Elife 9: e60827.
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