This protocol describes a method of live cell imaging using primary rat neonatal cardiomyocytes following lentiviral and adenoviral transduction using confocal spinning disk microscopy. This enables detailed observations of cellular processes in living cardiomyocytes.
Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single procedure. Due to advances in microscope technology it is relatively easy to capture live cell images for the purpose of investigating cellular events in real time with minimal concern regarding phototoxicity to the cells. This protocol describes how to take live cell timelapse images of primary rat neonatal cardiomyocytes using a confocal spinning disk microscope following lentiviral and adenoviral transduction to modulate properties of the cell. The application of two different types of viruses makes it easier to achieve an appropriate transduction rate and expression levels for two different genes. Well focused live cell images can be obtained using the microscope’s autofocus system, which maintains stable focus for long time periods. Applying this method, the functions of exogenously engineered proteins expressed in cultured primary cells can be analyzed. Additionally, this system can be used to examine the functions of genes through the use of siRNAs as well as of chemical modulators.
Primary rat neonatal cardiomyocytes have long been used for investigating cardiomyocyte function in vitro1. They are easy to isolate from rat pups by several well established methods2-4. The most common method employs collagenase or trypsin to digest connective tissue of the heart prior to cell isolation. Researchers have also developed methods for isolating cardiomyocytes from adult rodents5-8 as well as neonatal mice9,10.
This protocol describes a method for isolating cardiomyocytes from neonatal rat pups, employing a two-step enzyme digestion procedure. Trypsin is first used O/N at 4 °C, and then purified collagenase at 37 °C. Incubation of the heart tissue with trypsin O/N at 4 °C reduces the steps necessary to harvest cells compared to methods using sequential incubations in a warm enzyme solution2. In addition, by using purified collagenase rather than crude enzymes, lot-to-lot variability can be eliminated, thus providing enhanced reproducibility.
Functional studies of a particular protein often employ a protein expression system using an adenovirus11-13 and/or a lentivirus14-16. [CAUTIONARY NOTE] The viral production and manipulation should be carried out according to the NIH guidelines.
The adenovirus does not integrate into the host genome. It has a very high efficiency of transduction in most cell types, including dividing cells and non-dividing cells, as well as primary cells and established cell lines. This makes the adenovirus a reliable vector for gene expression. High levels of the protein encoded by the adenovirus vector develop within 48 hr following transduction, and they can last for several weeks17. However, one drawback to using an adenovirus for protein expression is that the development of a recombinant adenovirus is both complicated and time consuming. This drawback explains in part why many researchers have been turning to lentiviruses for recombinant gene expression. Unlike adenoviral constructs, generating a lentiviral construct is quick and easy. Although lentiviruses generally have lower efficiencies of transduction than adenoviruses, in both dividing and non-dividing cells, they do integrate into the host genome. Consequently, expression of the transduced gene is more stable for lentiviruses than for adenoviruses.
Due to technological advances in the field of microscopy, it is much easier to capture live cell images of cells expressing recombinant proteins. This holds true even at video rate speeds of acquisition. This allows the investigator to determine how particular alterations in the protein of interest functionally impact the cell in real time. The confocal spinning disk microscope has several key features that make it an optimal technique for live cell imaging18,19. The Yokogawa spinning disc allows for much more rapid image acquisition, while at the same time utilizing far less laser power than point scanning confocal microscopes. Both of these special features are due to the spinning disk, which contains numerous confocal holes through which the laser passes simultaneously to the samples. During acquisition, the disk itself spins quickly and continuously18-20. By using the autofocus system of the microscope, stable focus is maintained over long periods of time. This allows researchers to take well-focused live cell images. Acquired images are played back as a movie file. The images are analyzed using image analysis software such as ImageJ21,22, FIJI23, or other commercially available software.
1. Isolation of rat neonatal cardiomyocytes
Base medium | FBS | BrdU (mM) | P/S (U/ml) | |
selection | DMEM | 10% | 0 | 20 |
The first day | DMEM | 10% | 0.1 | 10 |
The next day | DMEM/MEM | 5% | 0.1 | 10 |
Further culture | DMEM/MEM | 5% | 0 | 10 |
Table 1: Medium for rat neonatal cardiomyocytes. Use this media for culturing rat neonatal cardiomyocytes. DMEM/MEM is 1:1 mix of DMEM and MEM medium.
1.1) Cardiomyocyte isolation, Day 1 (Estimated required time, about 1 hr)
NOTE: For work with neonatal rodents, refer to local university guidelines and rules set by animal care programs, and adhere to institutionally approved animal protocols. All methods described in this protocol have been approved by the Institutional Animal Care and Use Committee of the Yale Animal Resource Center.
1.2) Cardiomyocyte isolation, Day 2 (Estimated required time, about 4 hrs)
2. Lentiviral transduction
2.1) Packaging of lentiviral plasmids
NOTE: Please refer to other sources for further in-depth information on this subject 24-26. It will take about 3 days to prepare the lentiviral solution. It is best to use fresh lentiviral solution to achieve higher transduction efficiency. Start the packaging of lentiviral plasmids and isolation of rat neonatal cardiomyocytes in parallel. Instead of using polyethyleneimine (PEI) 27 for packaging of lentiviral plasmids, a commercially available transfection reagent can be used. Follow the manufacturer’s instructions.
Name of plasmid | Note | Size (kbp) | volume added (ml) | Final amount in 10 cm dish (mg) | Final concentration in 10 cm dish (pM) |
Lentiviral transfer vector | encodes gene to be packaged | 9-13 | 3.6-5.2 | 3.6-5.2 | 60 |
pMDLg/pRRE | expresses HIV-1 GAG/POL | 8.8 | 1.76 | 1.76 | 30 |
pRSV-Rev | expresses HIV-1 REV | 4.1 | 0.82 | 0.82 | 30 |
pMD2.G | expresses VSV G | 5.8 | 1.16 | 1.16 | 30 |
Table 2: The amount of plasmids for lentiviral packaging. Use these plasmid amounts to transfect HEK293 cells in 10 cm dishes. Final amount of lentiviral transfer vector per dish may differ according to its size, maintain final concentration per dish at 60 pM. Average molecular weight of one base pair of double stranded DNA is 660 daltons.
2.2) Collection of lentiviral solution and transduction
2.3) Concentration of lentiviral solution
NOTE: It is best to use fresh lentiviral solution in order to achieve highest transduction efficiencies, in case the lentiviral solution titer is not high enough, the lentiviral solution can be concentrated by polyethylene glycol (PEG) precipitation 29.
2.3.1) Preparation of 4x PEG solution (32% PEG6000, 400 mM sodium chloride (NaCl), 40 mM HEPES pH7.4)
2.3.2) Lentivirus concentration
3. Adenoviral transduction
NOTE: Please refer to other sources for methods for the construction and propagation of adenoviral constructs 13. Aspirating Pasteur pipettes and/or tips must be decontaminated by 10% bleach. Always decontaminate the biological safety cabinet surface and potentially contaminated equipment with 70% EtOH regardless of whether supernatant has been spilled. If spilling has occurred, thorough decontamination with 1% SDS in 70% EtOH is necessary.
lane | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
Row A | d | d | d | d | d | d | d | d | d | |||
Row B | d | d | d | d | d | d | d | d | d | |||
Row C | d | d | d | d | d | d | d | d | d | |||
Row D | d | d | d | d | d | d | d | |||||
Row E | d | d | d | d | d | d | d | d | d | d | d | |
Row F | d | d | d | d | d | d | d | |||||
Row G | d | d | d | d | d | d | d | d | ||||
Row H | d | d | d | d | d | d | d | d | d | d | d | |
the number of positive wells per row | 8 | 8 | 8 | 8 | 8 | 8 | 8 | 6 | 5 | 2 | 2 | 0 |
the ratio of positive wells per row | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0.75 | 0.63 | 0.25 | 0.25 | 0 |
d | Displaying over 50% cytopathic effects |
Displaying under 50% or no cytopathic effects |
Lane 1, dilution 31x104; lane 2, dilution 32x104; … ; lane 11, dilution 32x1011; lane 12, control.
S = the sum of the ratios of positive wells per row
= 1+1+1+1+1+1+1+0.75+0.63+0.25+0.25 +0= 8.875
TCID50 = 3 x 104 x 3 x (8.875-0.5) = 2.97 x 108
TCID50/ml = 5.94 x 109
Table 3: Example of an infected 96 well-plate after 10 days incubation. Measure the cytopathic effects in each well and sum the ratios of positive wells per row.
3.2) Adenoviral transduction
formula | example |
MOI = PFU of virus stock / number of cells | 100 (MOI) = 6 (ml) x 5.94 x 106 (TCID50/ml) x 0.56 / |
2 x 105 (cells) |
The PFU is proportional to the TCID50 with a factor of about 0.56.
Titer of virus stock for example = 5.94 x 109 (TCID50/ml) = 5.94 x 106 (TCID50/µl)
Example: Add 6 ml of virus stock with a titer of 5.94 x 109 (TCID50/ml) to a dish containing 2 x 105 cells to achieve an MOI of 100.
Table 4
MOI | Volume from stock (ml) | Volume to add serum free medium (ml) | Volume to add to dish (ml) |
1 | 3 | 197 | 2 |
3 | 3 | 197 | 6 |
10 | 3 | 197 | 20 |
30 | 3 | 197 | 60 |
100 | 6 | 194 | 200 |
Table 5
4. Live cell imaging
4.1) Image acquisition using a confocal spinning disk microscope with a temperature controlled chamber and a CO2 environmental system (optional)
4.2) Analysis of acquired images
NOTE: Acquired images can be played back as a movie file and analyzed using the analysis software.
To illustrate the technique, a lentivirus encoding EGFP (enhanced green fluorescent protein)-tagged Cx43 (Connexin43) or a mutated Cx43 32 was used to express EGFP-tagged proteins in cells, and an adenovirus encoding FGFR1DN (fibroblast growth factor receptor 1 dominant negative) was used to shut down FGF signaling in the cell 33-35. Three days following the isolation of cardiomyocytes, the isolated cardiomyocytes were transduced with lentivirus in order to express EGFP-tagged proteins in the cells. Then after 3 days of lentiviral transduction, the isolated cardiomyocytes were further transduced with an adenovirus encoding FGFR1DN to eliminate FGF signaling in the cells. After allowing enough time for cells to express the transduced exogenous genes, a confocal spinning disk microscope was used to capture live cell images. Acquired images are played back as a movie file and analyzed using image analysis software. For more detailed representative results please see Sakurai et al32.
The expression of EGFP tagged proteins following lentiviral transduction was confirmed by confocal spinning disk microscopy 72 hr following lentiviral transduction Figures 1A-B. The expression of FGFR1DN by adenoviral transduction was confirmed by WB and ICC after 24 hr of adenoviral transduction Figures 1C-D. The time of adenoviral addition to 3.5 cm dishes was defined as time 0 and time-lapse images were acquired with a 40x air NA 0.9 objective lens every 5 min for 6 hr using a chamber heater to maintain the temperature at 37 ˚C. Time-lapse images of EGFP-tagged proteins in the primary cardiomyocytes taken by confocal spinning disk microscopy are shown in Supplemental Movies 1-2.
In cases where the temperature equilibration was not adequate, or the multiple-acquisition mode was not functioning properly, images captured might move in the x-y plane Supplemental Movie 1. However when working properly the autofocus system stably maintained focus and this did not occur. As shown in Supplemental Movie 2, when the temperature equilibration was adequate and only one region of interest was acquired, the images captured are of very high quality.
The beating of cardiomyocytes was assessed after long-term time-lapse live cell imaging in order to confirm cell viability. Even after 16 hr of time-lapse imaging EGFP expressing cardiomyocytes maintained their functional properties and beat rhythmically Supplemental Movie 3.
Figure 1. Confirmation of protein expression following lentiviral or adenoviral transduction. (A) Isolated rat neonatal cardiomyocytes expressing Cx43-WT-EGFP transduced with Ad-FGFR1DN 3 days after transduction. See also Supplemental Movie 1. (B) Isolated rat neonatal cardiomyocytes expressing Cx43-S325/328/330E(3SE)-EGFP transduced with Ad-FGFR1DN 3 days after transduction. See also Supplemental Movie 2. (C) Protein expression following adenoviral transduction detected by ICC 24 hr after transduction. Hemagglutinin (HA) protein-tagged FGFR1DN was detected by anti-HA antibody using conventional ICC methods, alpha-actinin was used as a marker for cardiomyocytes. (D) Expression of FGFR1DN protein following adenoviral transduction detected by WB 24 hr after transduction. HA-tagged FGFR1DN was detected by anti-HA antibody by WB, beta-tubulin was used as loading control.
Supplemental Movie 1. Time-lapse imaging of isolated rat neonatal cardiomyocytes expressing Cx43-WT-EGFP transduced with Ad-FGFR1DN. The time-lapse images were acquired with a 40x objective lens every 5 min for 6 hr with a confocal spinning disk microscope. Acquired images were played back as a movie at 10 frames per sec. This movie has been modified from Sakurai et al32. Please click here to view this video.
Supplemental Movie 2. Time-lapse imaging of isolated rat neonatal cardiomyocytes expressing Cx43-S325/328/330E-EGFP transduced with Ad-FGFR1DN. The time-lapse images were acquired with a 40x objective lens every 5 min for 6 hr with a confocal spinning disk microscope. Acquired images were played back as a movie at 10 frames per sec. This movie has been modified from Sakurai et al32. Please click here to view this video.
Supplemental Movie 3. Time-lapse imaging of isolated rat neonatal cardiomyocytes played back at actual speed. The time-lapse images of isolated rat neonatal cardiomyocytes expressing Cx43-EGFP transduced with Ad-FGFR1DN were acquired with a 40x objective lens every 200 msec for 10 sec with a confocal spinning disk microscope following 16 hr of time-lapse acquisition. Acquired images were played back as a movie at 5 frames per sec at actual speed. Please click here to view this video.
Primary cardiomyocytes isolated from neonatal rats have long been used to study cardiomyocyte functions in vitro. This protocol describes a method for the isolation of neonatal cardiomyocytes from rat pups using a two-step enzyme digestion method, first digesting with trypsin O/N at 4 °C and then purified collagenase. One advantage of employing the purified collagenase step is that the same grade of enzyme is used for all isolations. Thus, the quality and amount of isolated cells is consistent from experiment to experiment.
Given the higher transduction efficiencies associated with adenovirus, if a high efficiency of transduction of the cell culture is necessary for a given experiment, it is better to use an adenovirus. However, if high levels of transduction are not necessary, a lentiviral vector can be used. Having the option of using either adenoviral or lentiviral vectors makes it easier to achieve appropriate transduction and expression levels for different types of genes. If the fluorescent intensity detected by live-cell imaging using the confocal spinning disk microscope or ICC is very low after lentiviral transduction, using another lentiviral transfer vector that uses a different promoter to express the gene of interest may resolve this problem. With adenoviral vectors, if the band detected by WB is weak after adenoviral transduction, this may indicate a low titer viral stock. In that case, it is better to prepare a higher titer adenoviral solution by propagation in cells before proceeding.
One limitation of this technique is that the yield of isolated viable cardiomyocytes is not highly consistent from experiment to experiment, even when using purified collagenase. In order to perform reproducible imaging experiments it is best to count viable cells after the selection of isolated cardiomyocytes in the supernatant at step 1.2.13. in the protocol section. Then, seed cardiomyocytes on glass bottom dishes at varying dilutions. Once the cells have attached, select dishes with the appropriate cell concentration for the experiment planned.
There are many advantages of using neonatal cardiomyocytes, but there are also some disadvantages. The major drawback is their clear difference from adult cardiomyocytes. Fully differentiated adult cardiomyocytes are rod-shape and isolated adult primary cardiomyocytes maintain their rod-shaped morphology, whereas isolated neonatal primary cardiomyocytes spread in all directions36. The phenotypic differences between isolated adult and neonatal cardiomyocytes are also reflected in their patterns of gene expression 37. Therefore, for experiments in which differentiated cardiomyocytes are required, a protocol for the isolation of adult cardiomyocytes will be needed4.
Employing adenoviral and lentiviral transduction of recombinant proteins into primary rat neonatal cardiomyocytes in combination with confocal spinning disk microscopy allows us to analyze the functions of proteins in cultured live primary cells. Relative to existing methods, which utilize transfection to introduce the gene of interest into cells, a significant advantage of this protocol is that adenoviruses and lentiviruses are very efficiently taken up by the cells resulting in the expression of the gene of interest in almost all cells in the culture. Furthermore, the use of two different types of viruses for gene expression makes it easier to achieve appropriate transduction rates and expression levels for multiple genes. siRNAs and chemical agonists and antagonists can also be used in this system to further perturb and analyze the functions of genes and the proteins they encode.
Preparation of the hearts is an extremely important step in the protocol. Remove the hearts from the body as quickly as possible. Be sure to put them on ice immediately to keep cell viability high. Another important step is the preparation of high quality, and titer, lentiviral/adenoviral stocks. If poor quality viral stocks are used, transduction and expression levels of the gene of interest will be low. Therefore, they may not be suitable for imaging studies.
An important future application of this technique will be its’ use in mouse cardiomyocytes. Pilot studies testing whether this method can be used for the isolation of mouse neonatal cardiomyocytes have yielded promising results. Due to the relatively small size of mouse hearts compared to those of rats, less tissue is obtained initially. Thus, fewer cells are isolated in a single procedure using the same number of animals but simply using more mice for the isolation should eliminate this obstacle. The ability to isolate mouse cardiomyocytes allows researchers to investigate the functions of cells isolated from genetically modified mice (transgenic, knock-out, knock-in) that have been produced in order to study a particular form of the gene of interest.
One of the major hurdles in cardiovascular research has been the difficulty associated with investigating the expression and function of a given gene expressed in heart tissue in vivo in real time. However the ability to isolate functional beating cardiomyocytes, modulate cell functions using viral transduction, and then study them in real time using the confocal spinning disk microscope helps to shed light on the role of cardiomyocytes in vivo and bridge this gap. This method provides a simplified system for understanding cardiomyocyte function at the cellular level. It allows for real time analysis of how perturbations in gene expression alter cardiomyocyte function. Live cell imaging of cardiomyocytes will provide further insights into cardiomyocyte function. Such insights will lead to advances in basic cardiovascular research, resulting in novel therapies for the treatment of cardiovascular disease.
The authors have nothing to disclose.
We would like to thank Dr. Alengo Nyamay’antu and Dr. Ilse Timmerman for their advices about lentiviral packaging. This work is supported by an American Heart Association Scientist Development Grant 10SDG4170137.
1 scissors for decapitation | WPI | 501749 | Autoclave before use |
1 fine scissors for heart isolation and chopping | WPI | 14393 | Autoclave before use |
2 fine forceps (Dumont No. 5) | Sigma | F6521 | Autoclave before use |
Three sterilized 10 cm plastic dishes | Sigma | CLS430165 | for hearts isolation |
3.5 cm glass bottom culture dishes | MatTek | P35G-1.5-20-C | for final plating of cardiomyocytes for future live cell imaging. micro-Dishes from ibidi are an acceptable alternative. |
3.5 cm glass bottom culture dishes | MatTek | P35G-0.17-14-C | for TIRF or high resolution image |
Ethanol solution, 70 % (v/v) in water | Sigma | E7148 | |
2% gelatin | Sigma | G1393 | |
One sterilized 10 cm plastic dish | Sigma | CLS430165 | for trypsinization |
Aluminium foil | any brand | ||
parafilm | Sigma | P7543 | |
Two 10 cm plastic cell culture dish | Sigma | CLS430165 | for selection |
Auto pipette | Drummond Scientific | 4-000-300 | for trituration |
Cell counter | |||
Cellometer, automated cell counter | nexcelom | to check and count cells | |
Microscope and Hematocytometer | any brand | to check and count cells | |
Trypan Blue Solution, 0.4% | invitrogen | 15250-061 | |
CO2 incubator | Sanyo | MCO-19AIC | |
incubating orbital shaker | Sigma | Z673129 | to shake heart tissue with collagenase at 37 C at 170-200 rpm |
10 mg/ml BrdU solution | BD Pharmingen | 550891 | |
DMEM, High Glucose | invitrogen | 41965039 | Mix medium as indicated in the protocol and warm before use |
MEM | invitrogen | 31095029 | Mix medium as indicated in the protocol and warm before use |
Fetal Bovine Serum | invitrogen | 26140079 | |
Penicillin-Streptomycin (10,000 U/mL) | invitrogen | 15140-122 | |
Section 2 lentiviral transduction | |||
three packaging plasmids | |||
pMDLg/pRRE | Addgene | 12251 | |
pRSV-Rev | Addgene | 12253 | |
pMD2.G | Addgene | 12259 | |
The lentiviral transfer vector, pLVX-IRES-Puro | Clontech | 632183 | |
Opti-MEM (serum-free medium) | invitrogen | 31985070 | |
transfection reagent | |||
polyethyleneimine“Max”, (Mw 40,000) – High Potency Linear PEI (Equivalent to Mw 25,000 in Free Base Form) | Polysciences | 24765-2 | It can be substituted with X-tremeGENE 9 from Roche |
X-tremeGENE 9 | Roche | 6365779001 | substitute for PEI as transfection reagent |
chloroquine | Sigma | C6628 | dissolve in water and make 100 mM stock solution. Inhibition of endosomal acidification can be achieved with 10-100 μM Chloroquine. |
HEPES | Sigma | H3375 | |
10 ml Luer-Lok syringe, sterilized | BD | 309604 | |
0.45 um filters | Sigma | F8677 | use only cellulose acetate or polyethersulfone (PES) (low protein binding) filters. Do not use nitrocellulose filters. Nitrocellulose binds surface proteins on the lentiviral envelope and destroys the virus. |
Hexadimethrine bromide | Sigma | H9268 | dissolve in water and make 8mg/ml stock solution, then filter it to sterilize. |
polyethylene glicol 6000 | Sigma | 81260 | |
Sodium chloride | Sigma | S9888 | |
sodium hydroxide | Sigma | S5881 | |
Section 3 Adenoviral transduction | |||
HEK 293T cells | ATCC | CRL-11268 | |
Some 10 cm cell culture dishes | Sigma | CLS430165 | |
96-Well Microplate with lid, flat-bottom, tissue culture, sterile | BD Falcon | 353072 | for titration |
Multichannel piptte, 10-100 ul, 8-channel | eppendorf | 3122 000.035 | |
Section 4 live cell imaging | |||
Spinning disk confocal microspopy | PerkinElmer | L7267000 | |
a temperature controlled chamber | any brand | to keep temperature at 37 C | |
a CO2 environmental system | any brand | optional to maintain CO2 concentration optimal | |
Medium | |||
CO2 Independent Medium, No Glutamine | invitrogen | 18045-054 | for long time time-lapse imaging |
DMEM, High Glucose, HEPES, no Phenol Red | invitrogen | 21063-029 | for long time time-lapse imaging |