Neonatal Cardiomyocyte Isolation System - Manual

Most traditional methods published for isolating neonatal cardiomyocytes utilize crude and partially purified enzyme preparations such as trypsin NF 1:250 (Gross, et al. 1968) and collagenase. More recently the use of better characterized preparations of collagenase such as Worthington type 2 collagenase (CLS2) has provided better, more reproducible results. Like all crude collagenase preparations, though there can be significant lot-to-lot variations so many researchers make use of the Worthington Sampling Program to pre-screen several lots of enzyme before selecting a lot to purchase. Pre-sampling assures that the enzyme preparation ultimately purchased will be satisfactory; however, it is time consuming.

The Worthington Neonatal Cardiomyocyte Isolation System has been introduced to provide researchers with a reliable, convenient, and consistent cell isolation system. By utilizing purified rather than crude enzyme preparations, it has been possible to minimize the lot-to-lot variation. In addition, Worthington use-tests the kits by isolating cardiomyocytes from neonatal rat hearts to assure performance, reliability, and consistent yield of viable cells.

The kit has been formulated in conjunction with Dr. Ronal MacGregor of the University of Kansas Medical Center. The method is based on that described by Toraason, et al. (1988) in which the minced tissue is incubated overnight with trypsin in the cold. As pointed out by Toraason, this step reduces the hands on time required to harvest cells compared to the time involved in sequential incubations in warm trypsin or collagenase.

Description and Package Contents:

The package contains sufficient materials for five separate tissue dissociations, each containing up to twelve hearts. For larger or smaller tissue samples prepare proportionate volumes of reagents at each step and combine them in the same ratio as described in the protocol.

Vial 1: 1 bottle, 500 ml

Sterile calcium- and magnesium-free Hank's Balanced Salt Solution (CMF HBSS), pH 7.4. The solution is used for reconstituting the contents of Vials #2 and #3 in addition to serving as the medium for the dissociation.
 

Vial 2: 5 vials, 1000 µg each

Worthington Trypsin (Code: TRLS), 3X crystallized, dialyzed against 1 mM HCl, filtered through 0.22 µm pore size membrane, and lyophilized. Before use, reconstitute with 2 ml CMF HBSS (Vial #1) and swirl gently to dissolve contents. Store at 2-8°C.
 

Vial 3: 5 vials, 2000µg each:

Worthington Soybean Trypsin Inhibitor (Code: SIC), a 0.22 micron pore size membrane filtered, lyophilized powder. Before use, reconstitute with 1ml CMF HBSS (Vial #1) and swirl gently to dissolve contents. Store at 2-8°C.
 

 

Vial 4: 5 vials, 1500 Units each

Worthington Purified Collagenase (Code: CLSPA), a 0.22 µm pore size membrane filtered, lyophilized powder which has been chromatographically purified. It contains less than 50 caseinase units per milligram and is composed of two separable but very similar collagenases. Before use, reconstitute with 5 ml Leibovitz L-15 Media (prepared as described below) and swirl gently to dissolve contents. Store at 2-8°C.
 

Pouch containing Leibovitz L-15 Media Powder: 1x1 L

Reconstitute entire contents of pouch by cutting open top of envelope and pouring contents into beaker containing 800 ml of cell culture grade water. Rinse pouch 2 - 3 times with additional 100 ml. Bring total volume to 1 liter and filter through a 0.22 micron filter.

 

The kit also includes 5 Cell Strainers (Falcon), and a card correlating phenol red color with pH for checking the pH of balanced salt solution and culture medium.

Needed but not supplied:

• Sterile 50 ml centrifuge tubes
• 10 cm Petri dishes, one per dispersion
• Wide-mouth 10 ml serological pipet with 3 mm diameter opening
• Standard 10 ml plastic serological pipet
• Rotating or shaking instrument for incubating at 37°C
• Oxygen supply
• Centrifuge (capable of 50-100 x g)
• Cultureware
• Sterile cell culture grade water
• Fetal Bovine Serum (FBS) for cell culture

For Cell Quantitation and Viability Assessment:

• Improved Neubauer Hemocytometer
• Counter
• Pasteur Pipet or Micropipettor
• Microscope (10X), preferably inverted phase-contrast
• Standard 10 ml serological pipets

Procedure

The volumes specified in the following protocol are suitable for hearts from 10 rat pups, 1 to 7 days old: Adjust proportionally for different numbers of hearts.

Note:

Do not process more than one litter or about 15 hearts in one Petri dish or 50 ml tube. The procedure works best for 5-15 hearts per preparation.

Note:

All glass and plasticware is sterile. Steps #1-4 may be performed in a cold room without ice, but a sterile hood is preferable.

Day 1: Perform the following in the afternoon

Prepare:

• Reagent #1, CMF HBSS: 50-60 ml from Vial #1, ice cold.
• Reagent #2, Trypsin: reconstitute one of Vial #2 with 2 ml Reagent #1, ice cold.
• One sterile 50 ml centrifuge tube, in ice.
• 10 cm Petri dish, sterile, on ice.

1. Transfer 30-40 ml of Reagent #1 to the centrifuge tube.
2. Anesthetize each rat pup, sterilize the abdomen with an antiseptic solution, and surgically remove the beating heart; immediately place the heart in the centrifuge tube to chill and rinse. Repeat for remaining rat pups. Swirl the tube to rinse hearts, then pour off most of the liquid. Rinse the hearts with 10 ml of Reagent #1, pour off the liquid as before, then transfer the hearts to the Petri dish. Mince the tissue with small scissors or a razor blade to less than 1 mm³ pieces keeping tissue at 0°C.
3. Add Reagent #1 to Petri dish to a final volume of approximately 9 ml.
4. Transfer 1 ml of the contents of the trypsin vial (Vial #2) into the Petri dish and mix completely by swirling. Final trypsin concentration is 50 µg/ml.
5. Place the lid on the petri dish and immediately place in refrigerator overnight (16-20 hours) at 2-8°C.

Note: If animals are 4 days old or older, increase the trypsin concentration up to a maximum of 100 µg/ml.

Day 2: Begin the following in the morning:

Prepare:

• Reagent #1, CMF HBSS: 30 ml. ice cold.
• Reagent #3, Trypsin Inhibitor: reconstitute one of Vial #3 with 1 ml Reagent #1. Room temperature.
• Reagent #4 Collagenase: reconstitute one of Vial #4 with 5 ml prepared Leibovitz L-15. Room temperature.
• Enough culture medium containing calcium and magnesium for digestion, centrifugations, and plating in cultureware. (approximately 100 ml for 10 hearts). Room temperature.
• Wide-mouth 10 ml serological pipet, sterile (opening about 3 mm diameter)
• Standard 10 ml plastic serological pipet

1. Remove Petri dish from refrigerator and bring to sterile hood on ice. Transfer tissue and buffer to 50 ml centrifuge tube on ice using wide-mouth pipet.
2. Transfer contents of Vial #3 into tube and mix.
3. Oxygenate tissue for 30 seconds to 1 minute if O₂ is available by passing oxygen over the surface of the liquid.
4. Warm tissue and buffer to 30-37°C in water bath, maintaining sterility (i.e. cap if needed). Do not add calcium-containing medium until tissue fragments are warm.
5. Slowly transfer the contents of Vial #4 into tube and mix. Cap tube tightly.
6. Place tube in/on slowly rotating (tumbling) or shaking instrument (2-4 rpm) at 37°C and incubate for 30 to 45 minutes.
   
   All subsequent steps at room temperature.
7. Remove tube from incubator and return to sterile hood. With standard 10 ml plastic serological pipet, triturate about 10 times to release cells. (Trituration is discussed in the following inset.) Pipet as gently as possible consistent with successful tissue dispersion.
8. Rinse a Cell Strainer with 1 ml of the L-15 culture medium. Allow tissue residue to settle 3-4 minutes, then (with same pipet) filter the supernatant through the Cell Strainer into a fresh 50 ml centrifuge tube.
9. Add 5 ml additional L-15 culture medium to tissue residue, repeat trituration step. Allow tissue residue to settle as before, then filter cells through the same Cell Strainer. Rinse mesh gently with 2 ml culture medium, oxygenate cells 1 minute, then allow filtered cells to remain undisturbed for about 20 minutes at room temperature. This allows complete digestion of the partially degraded collagen. (Cells can be held up to 1 hour at this point.)
10. Swirl cells gently; if no clumps have formed and appearance is uniform, sediment cells at 50-100 x g for 5 minutes (enough to settle the myocytes and some but not all red cells.) Suspend cells in additional portions of L-15 culture medium and repeat sedimentation as desired. If no sedimentation is desired, cells can be plated directly from the initial filtrate. Serum is generally required for plating cells in cultureware.
11. Suspend final cell pellet in suitable culture medium. Pipet gently to disperse. No clumps or connective tissue strands should be visible. Count the cells using a hemocytometer or other method, adjust cell concentration and add serum as desired, then dispense to tissue cultureware. (Some brands of uncoated cultureware do not encourage high plating efficiencies. Use Falcon or equivalant for best results.) Routine cell yields are 2-3 x 10⁶ cardiomyocytes per heart digested. Good (fairly heavy) seeding levels of cells should be obtained at 125,000 cardiomyocytes per cm² of culture wells or flasks. Adhesion may be improved by collagen or fibronectin coating of the plastic. Cell Quantitation and Estimation of Viability are discussed in the following sections.
12. Place each plate or flask in a 37°C incubator as soon as it is plated. Do not touch or otherwise disturb the cells for at least 24 hours.