Application of Three-Dimensional Culture Method in the Cardiac Conduction System Research

Congenital heart defects (CHD) are the most common type of birth defects. Several human case studies and genetically altered animal models have identified abnormalities in the development of ventricular conduction system (VCS) in the heart. While cell-based therapies hold promise for treating CHDs, translational efforts are limited by the lack of suitable in vitro models for feasibility and safety studies. A better understanding of cell differentiation pathways can lead to development of cell-based therapies for individuals living with CHD/VCS disorders. Here, we describe a new and reproducible 3-D cell culture method for studying cardiac cell lineage differentiation in vitro. We used primary ventricular cells isolated from embryonic day 11.5 (E11.5) mouse embryos, which can differentiate into multiple cardiac cell types including VCS cells. We compared 3-D cultures with three types of basement membrane extracts (BME) for their abilities to support E11.5 ventricular cell differentiation. In addition, the effects of atrial natriuretic peptide (ANP) and an inhibitor for its high affinity receptor were tested on cell differentiation in 3-D cultures. Following the cell culture, protocols for immunofluorescence imaging, cell extraction and protein isolation from the 3-D culture matrix and in-cell western methods are described. Further, these approaches can be used to study the effects of various ligands and genetic interventions on VCS cell development. We propose that these methodologies may also be extended for differentiation studies using other sources of stem cells such as induced pluripotent stem cells.


Introduction
A diverse array of genetic and structure-function changes has been implicated in the etiology of congenital heart defects (CHDs) and cardiac conduction system disorders [1][2][3]. While the outlook for patients living with these disorders is much better with improvements in surgical and medical management, these patients tend to develop arrhythmias and heart failure [4]. Advances in cardiac development and stem cell fields hold promise for the treatment of CHDs and conduction system abnormalities [5].
In mouse embryos, ventricular conduction system (VCS) development begins after embryonic day 11.5 (E11.5) and a fully functional VCS appears after E17.5 [6,7]. The gap junction protein, connexin 40 (Cx40) expression is uniform in the trabecular myocardium at E11 stage and it becomes restricted to the VCS and atria by E16 through neonatal stages [8,9]. There is evidence that factors secreted from the coronary endothelium, endocardium, and trabecular myocardium (e.g., endothelin-1, neuregulin-1 and atrial natriuretic peptide) provide instructive cues for the VCS cell fate [10][11][12][13]. Although two-dimensional (2-D) cultures of embryonic ventricular cells and embryonic stem cells have been used to study cell proliferation and differentiation patterns [14][15][16][17][18], mechanisms regulating VCS cell formation are not very well characterized. Notably, Purkinje fibers form a complex network of peripheral VCS components from base to apex in the subendocardial regions of the mammalian heart [19]. Development of novel cell culture models which permit formation CRITICAL STEP: Frequent thawing of the matrix can lead to loss of polymerization efficiency. To avoid multiple freeze thaw cycles, the stock solution of BME matrix can be aliquoted into multiple tubes and stored at −80 °C. This procedure must be performed on the ice platform or any chilled surface. All tubes and pipette tips must be pre-chilled and placed on ice during aliquot preparation.
PAUSE STEP: All the aliquoted tubes must be stored at −80 °C immediately for future use.

Thawing of BME Matrix
CRITICAL STEP: BME matrix is thawed overnight in the 4 °C environment prior to starting a 3-D culture experiment. Because refrigeration temperatures may vary, the BME aliquots should be kept on ice in the refrigerator during the thawing process.
PAUSE STEP: Thawed BME polymerizes rapidly at temperatures above 15 °C. It is important to keep it on ice to prevent its solidification prior to coating culture vessels. Pre-chilled pipette tips and tubes must be used to prevent untimely gelling of BME during the coating procedure.

Embryonic Ventricular Cell Isolation
 Embryos (E11.5) from the timed-pregnant CD1 mice (Charles River Laboratories, Senneville, Quebec, QC, Canada) are taken out from the uterus by removing the placenta and kept in dishes containing PBS with 1× Ab/Am. The whole heart is removed from the embryo, and then the atria and outflow tracts are dissected out.  Both ventricles from each embryonic heart are placed in 0.2% v/v type I Collagenase in 1× PBS (~10 ventricles in 1 mL) and incubated for 45 min at 37 °C on a rocking platform and the ventricular tissue is digested.
CRITICAL STEP: Collagenase digestion of E11.5 ventricles should not exceed 45 min for optimal cell viability and recovery.
CRITICAL STEP: After 45 min of incubation, the tissue is triturated using a pipette tip for mechanical dissociation of the remaining tissue. Subsequently, dissociated tissue fragments are allowed to settle for 5 min for gravity separation of the fibrous tissue debris.


The supernatant is recovered, leaving debris in the tube, and the aqueous fraction containing cells is centrifuged at 1600 rcf (Eppendorf centrifuge, model#5415D) for 4 min at 4 °C. The supernatant is discarded, and the cell pellet is neutralized by two washes of culture media (DMEM containing 10% FBS and 1× Ab/Am).  Finally, cells are resuspended using 10% FBS-DMEM, and the cell number is determined using a hemocytometer. The cell suspension is resuspended in media to achieve desired cell density for 3-D cultures.
CRITICAL STEP: Frequent thawing of the matrix can lead to loss of polymerization efficiency. To avoid multiple freeze thaw cycles, the stock solution of BME matrix can be aliquoted into multiple tubes and stored at −80 • C. This procedure must be performed on the ice platform or any chilled surface. All tubes and pipette tips must be pre-chilled and placed on ice during aliquot preparation.
Methods Protoc. 2022, 5, x FOR PEER REVIEW 5 of 17 PAUSE STEP: Dispersed cells can be stored on ice for 30 min to 2 h before plating on BME matrix. However, it is recommended to plate cells immediately after isolation.


Coat pre-chilled culture surface of 8-chambered slide with a thick layer of BME matrix in a sterile environment. The volume of the matrix needs to be optimized, but 60 µ L/chamber is a good starting point. Pipette the matrix in the center of the chamber and spread uniformly over the surface using a pre-chilled pipette tip. Carefully spread the matrix in all the corners of the chambered slide.
OPTIONAL STEP: 8-Chambered culture slides and pipette tips can be kept in −20 °C for pre-chilling few hours before the experiment.
CRITICAL STEP: BME matrix polymerizes and solidifies rapidly at room temperature. Working over ice platform or any chilled platform in the culture hood delays the polymerization process and makes the uniform spread of the matrix in chambers effortless. Uniform spreading of the matrix is a critical factor in this method. Uneven matrix bed can lead to cell accumulation in areas with a thin layer of matrix.
Note: Matrix provides an optimal milieu for fibers to grow in any direction. The thickness of the matrix bed is a regulatory factor in the formation of cardiac fibers. A very thin matrix bed does not support fiber formation; it instead leads to formation of cell clusters without connections. A thick matrix bed promotes a good cardiac fiber structure formation. However, a thick matrix bed presents some practical difficulties such as mounting a coverslip after the staining procedure and poor visualization under the higher magnification using a confocal microscope. The thickness of the matrix bed can be decided based on the procedures to be done after culture. For protein extraction and RNA isolation procedures, the thickness should be more (3-5 mm), and for immunostaining purposes, the thickness should be optimally less (1-2 mm).


Let the coated surface dry for 15-30 min at 37 °C in the incubator but avoid overdrying to prevent dehydration.  In the meantime, resuspend 125,000-250,000 cells in 100 µ L of culture media (DMEM-10%FBS) per chamber. This volume is half of the complete media volume to be put in one chamber.  After incubation of the pre-coated culture surface, pipette the mixture of embryonic ventricular cells and media over the matrix bed and incubate for 30 min in a cell culture incubator at 37 °C and 5% CO2.  Chill the remaining media on ice and mix 10% volume of the matrix (100 µ L media + 10 µ L matrix/chamber). Gently mix this mixture homogeneously and pipette over the incubated culture along the wall of the chamber.  Incubate the culture at 37 °C and 5% CO2 in the incubator. Depending on the experiment, treat the culture with desired drugs either during seeding of cells or a day after when the 3-D structure begins to form.  Monitor the growth of culture under an inverted phase-contrast microscope and observe for the formation of three-dimensional structures ( Figure 1C-G). Cell clusters formation starts in 2-3 days and connecting extensions between the contractile spheroid cell bodies can be observed in 7-10 days ( Figure 1A-G). A healthy culture can be maintained for 13-15 days. CRITICAL STEP: Frequent thawing of the matrix can lead to loss of polymerization efficiency. To avoid multiple freeze thaw cycles, the stock solution of BME matrix can be aliquoted into multiple tubes and stored at −80 °C. This procedure must be performed on the ice platform or any chilled surface. All tubes and pipette tips must be pre-chilled and placed on ice during aliquot preparation.
PAUSE STEP: All the aliquoted tubes must be stored at −80 °C immediately for future use.

Thawing of BME Matrix
CRITICAL STEP: BME matrix is thawed overnight in the 4 °C environment prior to starting a 3-D culture experiment. Because refrigeration temperatures may vary, the BME aliquots should be kept on ice in the refrigerator during the thawing process.
PAUSE STEP: Thawed BME polymerizes rapidly at temperatures above 15 °C. It is important to keep it on ice to prevent its solidification prior to coating culture vessels. Pre-chilled pipette tips and tubes must be used to prevent untimely gelling of BME during the coating procedure.

Embryonic Ventricular Cell Isolation 
Embryos (E11.5) from the timed-pregnant CD1 mice (Charles River Laboratories, Senneville, Quebec, QC, Canada) are taken out from the uterus by removing the placenta and kept in dishes containing PBS with 1× Ab/Am. The whole heart is removed from the embryo, and then the atria and outflow tracts are dissected out.  Both ventricles from each embryonic heart are placed in 0.2% v/v type I Collagenase in 1× PBS (~10 ventricles in 1 mL) and incubated for 45 min at 37 °C on a rocking platform and the ventricular tissue is digested.
CRITICAL STEP: Collagenase digestion of E11.5 ventricles should not exceed 45 min for optimal cell viability and recovery.
CRITICAL STEP: After 45 min of incubation, the tissue is triturated using a pipette tip for mechanical dissociation of the remaining tissue. Subsequently, dissociated tissue fragments are allowed to settle for 5 min for gravity separation of the fibrous tissue debris.  The supernatant is recovered, leaving debris in the tube, and the aqueous fraction containing cells is centrifuged at 1600 rcf (Eppendorf centrifuge, model#5415D) for 4 min at 4 °C. The supernatant is discarded, and the cell pellet is neutralized by two washes of culture media (DMEM containing 10% FBS and 1× Ab/Am).  Finally, cells are resuspended using 10% FBS-DMEM, and the cell number is determined using a hemocytometer. The cell suspension is resuspended in media to achieve desired cell density for 3-D cultures.
CRITICAL STEP: BME matrix is thawed overnight in the 4 • C environment prior to starting a 3-D culture experiment. Because refrigeration temperatures may vary, the BME aliquots should be kept on ice in the refrigerator during the thawing process.
Methods Protoc. 2022, 5, x FOR PEER REVIEW 5 of 17 PAUSE STEP: Dispersed cells can be stored on ice for 30 min to 2 h before plating on BME matrix. However, it is recommended to plate cells immediately after isolation.


Coat pre-chilled culture surface of 8-chambered slide with a thick layer of BME matrix in a sterile environment. The volume of the matrix needs to be optimized, but 60 µ L/chamber is a good starting point. Pipette the matrix in the center of the chamber and spread uniformly over the surface using a pre-chilled pipette tip. Carefully spread the matrix in all the corners of the chambered slide.
OPTIONAL STEP: 8-Chambered culture slides and pipette tips can be kept in −20 °C for pre-chilling few hours before the experiment.
CRITICAL STEP: BME matrix polymerizes and solidifies rapidly at room temperature. Working over ice platform or any chilled platform in the culture hood delays the polymerization process and makes the uniform spread of the matrix in chambers effortless. Uniform spreading of the matrix is a critical factor in this method. Uneven matrix bed can lead to cell accumulation in areas with a thin layer of matrix.
Note: Matrix provides an optimal milieu for fibers to grow in any direction. The thickness of the matrix bed is a regulatory factor in the formation of cardiac fibers. A very thin matrix bed does not support fiber formation; it instead leads to formation of cell clusters without connections. A thick matrix bed promotes a good cardiac fiber structure formation. However, a thick matrix bed presents some practical difficulties such as mounting a coverslip after the staining procedure and poor visualization under the higher magnification using a confocal microscope. The thickness of the matrix bed can be decided based on the procedures to be done after culture. For protein extraction and RNA isolation procedures, the thickness should be more (3-5 mm), and for immunostaining purposes, the thickness should be optimally less (1-2 mm).


Let the coated surface dry for 15-30 min at 37 °C in the incubator but avoid overdrying to prevent dehydration.  In the meantime, resuspend 125,000-250,000 cells in 100 µ L of culture media (DMEM-10%FBS) per chamber. This volume is half of the complete media volume to be put in one chamber.  After incubation of the pre-coated culture surface, pipette the mixture of embryonic ventricular cells and media over the matrix bed and incubate for 30 min in a cell culture incubator at 37 °C and 5% CO2.  Chill the remaining media on ice and mix 10% volume of the matrix (100 µ L media + 10 µ L matrix/chamber). Gently mix this mixture homogeneously and pipette over the incubated culture along the wall of the chamber.
PAUSE STEP: Thawed BME polymerizes rapidly at temperatures above 15 • C. It is important to keep it on ice to prevent its solidification prior to coating culture vessels. Pre-chilled pipette tips and tubes must be used to prevent untimely gelling of BME during the coating procedure.

Embryonic Ventricular Cell Isolation
• Embryos (E11.5) from the timed-pregnant CD1 mice (Charles River Laboratories, Senneville, Quebec, QC, Canada) are taken out from the uterus by removing the placenta and kept in dishes containing PBS with 1× Ab/Am. The whole heart is removed from the embryo, and then the atria and outflow tracts are dissected out.

•
Both ventricles from each embryonic heart are placed in 0.2% v/v type I Collagenase in 1× PBS (~10 ventricles in 1 mL) and incubated for 45 min at 37 • C on a rocking platform and the ventricular tissue is digested.

Storage and Handling of BME Matrix
3.1.1. Aliquoting and Freezing of BME Matrix CRITICAL STEP: Frequent thawing of the matrix can lead to loss of polymerization efficiency. To avoid multiple freeze thaw cycles, the stock solution of BME matrix can be aliquoted into multiple tubes and stored at −80 °C. This procedure must be performed on the ice platform or any chilled surface. All tubes and pipette tips must be pre-chilled and placed on ice during aliquot preparation.
PAUSE STEP: All the aliquoted tubes must be stored at −80 °C immediately for future use.

Thawing of BME Matrix
CRITICAL STEP: BME matrix is thawed overnight in the 4 °C environment prior to starting a 3-D culture experiment. Because refrigeration temperatures may vary, the BME aliquots should be kept on ice in the refrigerator during the thawing process.
PAUSE STEP: Thawed BME polymerizes rapidly at temperatures above 15 °C. It is important to keep it on ice to prevent its solidification prior to coating culture vessels. Pre-chilled pipette tips and tubes must be used to prevent untimely gelling of BME during the coating procedure.

Embryonic Ventricular Cell Isolation 
Embryos (E11.5) from the timed-pregnant CD1 mice (Charles River Laboratories, Senneville, Quebec, QC, Canada) are taken out from the uterus by removing the placenta and kept in dishes containing PBS with 1× Ab/Am. The whole heart is removed from the embryo, and then the atria and outflow tracts are dissected out.  Both ventricles from each embryonic heart are placed in 0.2% v/v type I Collagenase in 1× PBS (~10 ventricles in 1 mL) and incubated for 45 min at 37 °C on a rocking CRITICAL STEP: Collagenase digestion of E11.5 ventricles should not exceed 45 min for optimal cell viability and recovery.

Storage and Handling of BME Matrix
3.1.1. Aliquoting and Freezing of BME Matrix CRITICAL STEP: Frequent thawing of the matrix can lead to loss of polymerization efficiency. To avoid multiple freeze thaw cycles, the stock solution of BME matrix can be aliquoted into multiple tubes and stored at −80 °C. This procedure must be performed on the ice platform or any chilled surface. All tubes and pipette tips must be pre-chilled and placed on ice during aliquot preparation.
PAUSE STEP: All the aliquoted tubes must be stored at −80 °C immediately for future use.

Thawing of BME Matrix
CRITICAL STEP: BME matrix is thawed overnight in the 4 °C environment prior to starting a 3-D culture experiment. Because refrigeration temperatures may vary, the BME aliquots should be kept on ice in the refrigerator during the thawing process.
PAUSE STEP: Thawed BME polymerizes rapidly at temperatures above 15 °C. It is important to keep it on ice to prevent its solidification prior to coating culture vessels. Pre-chilled pipette tips and tubes must be used to prevent untimely gelling of BME during the coating procedure.

Embryonic Ventricular Cell Isolation 
Embryos (E11.5) from the timed-pregnant CD1 mice (Charles River Laboratories, Senneville, Quebec, QC, Canada) are taken out from the uterus by removing the placenta and kept in dishes containing PBS with 1× Ab/Am. The whole heart is removed CRITICAL STEP: After 45 min of incubation, the tissue is triturated using a pipette tip for mechanical dissociation of the remaining tissue. Subsequently, dissociated tissue fragments are allowed to settle for 5 min for gravity separation of the fibrous tissue debris.

•
The supernatant is recovered, leaving debris in the tube, and the aqueous fraction containing cells is centrifuged at 1600 rcf (Eppendorf centrifuge, model#5415D) for 4 min at 4 • C. The supernatant is discarded, and the cell pellet is neutralized by two washes of culture media (DMEM containing 10% FBS and 1× Ab/Am). • Finally, cells are resuspended using 10% FBS-DMEM, and the cell number is determined using a hemocytometer. The cell suspension is resuspended in media to achieve desired cell density for 3-D cultures.
Methods Protoc. 2022, 5, x FOR PEER REVIEW 5 of 17 PAUSE STEP: Dispersed cells can be stored on ice for 30 min to 2 h before plating on BME matrix. However, it is recommended to plate cells immediately after isolation.


Coat pre-chilled culture surface of 8-chambered slide with a thick layer of BME matrix in a sterile environment. The volume of the matrix needs to be optimized, but 60 µ L/chamber is a good starting point. Pipette the matrix in the center of the chamber and spread uniformly over the surface using a pre-chilled pipette tip. Carefully spread the matrix in all the corners of the chambered slide.
OPTIONAL STEP: 8-Chambered culture slides and pipette tips can be kept in −20 PAUSE STEP: Dispersed cells can be stored on ice for 30 min to 2 h before plating on BME matrix. However, it is recommended to plate cells immediately after isolation.

•
Coat pre-chilled culture surface of 8-chambered slide with a thick layer of BME matrix in a sterile environment. The volume of the matrix needs to be optimized, but 60 µL/chamber is a good starting point. Pipette the matrix in the center of the chamber and spread uniformly over the surface using a pre-chilled pipette tip. Carefully spread the matrix in all the corners of the chambered slide.
OPTIONAL STEP: 8-Chambered culture slides and pipette tips can be kept in −20 • C for pre-chilling few hours before the experiment.

Storage and Handling of BME Matrix
3.1.1. Aliquoting and Freezing of BME Matrix CRITICAL STEP: Frequent thawing of the matrix can lead to loss of polymerization efficiency. To avoid multiple freeze thaw cycles, the stock solution of BME matrix can be aliquoted into multiple tubes and stored at −80 °C. This procedure must be performed on the ice platform or any chilled surface. All tubes and pipette tips must be pre-chilled and placed on ice during aliquot preparation.
PAUSE STEP: All the aliquoted tubes must be stored at −80 °C immediately for future use.

Thawing of BME Matrix
CRITICAL STEP: BME matrix is thawed overnight in the 4 °C environment prior to starting a 3-D culture experiment. Because refrigeration temperatures may vary, the BME aliquots should be kept on ice in the refrigerator during the thawing process.
PAUSE STEP: Thawed BME polymerizes rapidly at temperatures above 15 °C. It is important to keep it on ice to prevent its solidification prior to coating culture vessels. Pre-chilled pipette tips and tubes must be used to prevent untimely gelling of BME during the coating procedure.

Embryonic Ventricular Cell Isolation
 Embryos (E11.5) from the timed-pregnant CD1 mice (Charles River Laboratories, Senneville, Quebec, QC, Canada) are taken out from the uterus by removing the placenta and kept in dishes containing PBS with 1× Ab/Am. The whole heart is removed from the embryo, and then the atria and outflow tracts are dissected out.  Both ventricles from each embryonic heart are placed in 0.2% v/v type I Collagenase in 1× PBS (~10 ventricles in 1 mL) and incubated for 45 min at 37 °C on a rocking platform and the ventricular tissue is digested.
CRITICAL STEP: Collagenase digestion of E11.5 ventricles should not exceed 45 min for optimal cell viability and recovery.
CRITICAL STEP: After 45 min of incubation, the tissue is triturated using a pipette tip for mechanical dissociation of the remaining tissue. Subsequently, dissociated tissue fragments are allowed to settle for 5 min for gravity separation of the fibrous tissue debris.


The supernatant is recovered, leaving debris in the tube, and the aqueous fraction containing cells is centrifuged at 1600 rcf (Eppendorf centrifuge, model#5415D) for 4 min at 4 °C. The supernatant is discarded, and the cell pellet is neutralized by two washes of culture media (DMEM containing 10% FBS and 1× Ab/Am).  Finally, cells are resuspended using 10% FBS-DMEM, and the cell number is determined using a hemocytometer. The cell suspension is resuspended in media to achieve desired cell density for 3-D cultures.
CRITICAL STEP: BME matrix polymerizes and solidifies rapidly at room temperature. Working over ice platform or any chilled platform in the culture hood delays the polymerization process and makes the uniform spread of the matrix in chambers effortless. Uniform spreading of the matrix is a critical factor in this method. Uneven matrix bed can lead to cell accumulation in areas with a thin layer of matrix.
Note: Matrix provides an optimal milieu for fibers to grow in any direction. The thickness of the matrix bed is a regulatory factor in the formation of cardiac fibers. A very thin matrix bed does not support fiber formation; it instead leads to formation of cell clusters without connections. A thick matrix bed promotes a good cardiac fiber structure formation. However, a thick matrix bed presents some practical difficulties such as mounting a coverslip after the staining procedure and poor visualization under the higher magnification using a confocal microscope. The thickness of the matrix bed can be decided based on the procedures to be done after culture. For protein extraction and RNA isolation procedures, the thickness should be more (3-5 mm), and for immunostaining purposes, the thickness should be optimally less (1-2 mm).

•
Let the coated surface dry for 15-30 min at 37 • C in the incubator but avoid over-drying to prevent dehydration.

•
In the meantime, resuspend 125,000-250,000 cells in 100 µL of culture media (DMEM-10%FBS) per chamber. This volume is half of the complete media volume to be put in one chamber.

•
After incubation of the pre-coated culture surface, pipette the mixture of embryonic ventricular cells and media over the matrix bed and incubate for 30 min in a cell culture incubator at 37 • C and 5% CO 2 .

•
Chill the remaining media on ice and mix 10% volume of the matrix (100 µL media + 10 µL matrix/chamber). Gently mix this mixture homogeneously and pipette over the incubated culture along the wall of the chamber.

•
Incubate the culture at 37 • C and 5% CO 2 in the incubator. Depending on the experiment, treat the culture with desired drugs either during seeding of cells or a day after when the 3-D structure begins to form.

•
Monitor the growth of culture under an inverted phase-contrast microscope and observe for the formation of three-dimensional structures ( Figure 1C-G). Cell clusters formation starts in 2-3 days and connecting extensions between the contractile spheroid cell bodies can be observed in 7-10 days ( Figure 1A-G To avoid multiple freeze thaw cycles, the stock solution of BME matrix can be aliquoted into multiple tubes and stored at −80 °C. This procedure must be performed on the ice platform or any chilled surface. All tubes and pipette tips must be pre-chilled and placed on ice during aliquot preparation. PAUSE STEP: All the aliquoted tubes must be stored at −80 °C immediately for future use. In the meantime, resuspend the isolated embryonic ventricular cells into an appro-  (Table 1). Depending on the half-life of drugs, the feeding protocol can be modified, but a long gap between feedings is harmful to cells.

•
Drugs are dissolved in 200 µL media per chamber, and caution must be taken while feeding the culture. The old media is aspirated out from each chamber slowly, and fresh media is added along the wall of the chamber to prevent the damage to the 3-D cultured matrix. ± ANP (1 µg/mL) and A71915 or A7 (1µM) for Figure 2 ± ANP (1 µg/mL) and A71915 or A7 (1µM) for Figure 2 ± ANP (1 µg/mL) and A71915 or A7 (1µM) for Figure 2 No drug addition CRITICAL STEP: Frequent thawing of the matrix can lead to loss of polymerization efficiency. To avoid multiple freeze thaw cycles, the stock solution of BME matrix can be aliquoted into multiple tubes and stored at −80 °C. This procedure must be performed on the ice platform or any chilled surface. All tubes and pipette tips must be pre-chilled and placed on ice during aliquot preparation.
PAUSE STEP: All the aliquoted tubes must be stored at −80 °C immediately for future use.

Thawing of BME Matrix
CRITICAL STEP: BME matrix is thawed overnight in the 4 °C environment prior to starting a 3-D culture experiment. Because refrigeration temperatures may vary, the BME aliquots should be kept on ice in the refrigerator during the thawing process.
PAUSE STEP: Thawed BME polymerizes rapidly at temperatures above 15 °C. It is important to keep it on ice to prevent its solidification prior to coating culture vessels. Pre-chilled pipette tips and tubes must be used to prevent untimely gelling of BME during the coating procedure.

Embryonic Ventricular Cell Isolation
 Embryos (E11.5) from the timed-pregnant CD1 mice (Charles River Laboratories, Senneville, Quebec, QC, Canada) are taken out from the uterus by removing the placenta and kept in dishes containing PBS with 1× Ab/Am. The whole heart is removed from the embryo, and then the atria and outflow tracts are dissected out.  Both ventricles from each embryonic heart are placed in 0.2% v/v type I Collagenase in 1× PBS (~10 ventricles in 1 mL) and incubated for 45 min at 37 °C on a rocking platform and the ventricular tissue is digested.
CRITICAL STEP: Collagenase digestion of E11.5 ventricles should not exceed 45 min for optimal cell viability and recovery.
CRITICAL STEP: After 45 min of incubation, the tissue is triturated using a pipette tip for mechanical dissociation of the remaining tissue. Subsequently, dissociated tissue fragments are allowed to settle for 5 min for gravity separation of the fibrous tissue debris.


The supernatant is recovered, leaving debris in the tube, and the aqueous fraction containing cells is centrifuged at 1600 rcf (Eppendorf centrifuge, model#5415D) for 4 min at 4 °C. The supernatant is discarded, and the cell pellet is neutralized by two washes of culture media (DMEM containing 10% FBS and 1× Ab/Am).  Finally, cells are resuspended using 10% FBS-DMEM, and the cell number is determined using a hemocytometer. The cell suspension is resuspended in media to achieve desired cell density for 3-D cultures.

CRITICAL STEP:
The polymerized matrix is very fragile in nature. A slight disturbance can break the matrix and spoil the 3-D culture. Pipetting in and out must be very slow and along the wall of the chamber. Pipetting from one corner of the chamber in a slightly tilted position of culture slide reduces the chances of rupture of the matrix. Multiple pipetting events in every step from the beginning of culture to downstream processing of 3-D culture product makes pipetting a very critical procedure.

•
Culture is fed until the desired 3-D culture morphology is achieved; 7 to 8 days are ideal for downstream procedures such as culture fixation for immunostaining (see

Grading of Spheroid Cell Bodies by Fiber Formation Analysis
• Fiber formation can be analyzed based on the number of outgrowths or fibers arising from each spheroid cell body in E11. 5

ventricular cells grown in 3-D cultures. •
Mean fiber numbers can be determined by blinded analysis of digital images captured using a 20× phase contrast objective from 6-7 random fields/group in each experiment. • Differences in fiber formation and distribution can be quantified by comparing the mean fiber numbers of spheroid cell bodies grown in 3-D cultures treated with different exogenous agonists and antagonists.
OPTIONAL STEP: The fiber scoring system is a subjective method to analyze the differences in the fiber formation in 3-D cultures. Scoring assignment can also be done as per the individual's need to determine differences between groups. Other factors can also be taken in consideration to analyze any differences in fiber formation, such as fiber connections between two cell bodies, number of collateral branches arising from each outgrowth and beating activities of fibers.

•
Aspirate the media slowly from the chamber and rinse the culture with an equivalent volume of PBS at room temperature. • Culture can be fixed by 4% paraformaldehyde (PFA) at room temperature.

Aliquoting and Freezing of BME Matrix
CRITICAL STEP: Frequent thawing of the matrix can lead to loss of polymerization efficiency. To avoid multiple freeze thaw cycles, the stock solution of BME matrix can be aliquoted into multiple tubes and stored at −80 °C. This procedure must be performed on the ice platform or any chilled surface. All tubes and pipette tips must be pre-chilled and placed on ice during aliquot preparation.
PAUSE STEP: All the aliquoted tubes must be stored at −80 °C immediately for future use.

Thawing of BME Matrix
CRITICAL STEP: BME matrix is thawed overnight in the 4 °C environment prior to starting a 3-D culture experiment. Because refrigeration temperatures may vary, the BME aliquots should be kept on ice in the refrigerator during the thawing process.
PAUSE STEP: Thawed BME polymerizes rapidly at temperatures above 15 °C. It is important to keep it on ice to prevent its solidification prior to coating culture vessels. Pre-chilled pipette tips and tubes must be used to prevent untimely gelling of BME during the coating procedure.

Embryonic Ventricular Cell Isolation
 Embryos (E11.5) from the timed-pregnant CD1 mice (Charles River Laboratories, Senneville, Quebec, QC, Canada) are taken out from the uterus by removing the placenta and kept in dishes containing PBS with 1× Ab/Am. The whole heart is removed from the embryo, and then the atria and outflow tracts are dissected out.  Both ventricles from each embryonic heart are placed in 0.2% v/v type I Collagenase in 1× PBS (~10 ventricles in 1 mL) and incubated for 45 min at 37 °C on a rocking platform and the ventricular tissue is digested.
CRITICAL STEP: Collagenase digestion of E11.5 ventricles should not exceed 45 min for optimal cell viability and recovery.
CRITICAL STEP: After 45 min of incubation, the tissue is triturated using a pipette tip for mechanical dissociation of the remaining tissue. Subsequently, dissociated tissue fragments are allowed to settle for 5 min for gravity separation of the fibrous tissue debris.


The supernatant is recovered, leaving debris in the tube, and the aqueous fraction containing cells is centrifuged at 1600 rcf (Eppendorf centrifuge, model#5415D) for 4 min at 4 °C. The supernatant is discarded, and the cell pellet is neutralized by two washes of culture media (DMEM containing 10% FBS and 1× Ab/Am).  Finally, cells are resuspended using 10% FBS-DMEM, and the cell number is determined using a hemocytometer. The cell suspension is resuspended in media to CRITICAL STEP: Fixation with cold 4% PFA can lead to the loss of matrix integrity, and loss of 3-D cell structures. Therefore, fixation solution should be prewarmed to the room temperature.

•
After 10 min of fixation at room temperature, treat the fixed cultures with PBS-glycine for 5-10 min to decrease non-specific binding during immunofluorescence staining. Wash the culture with PBS and then process for immunostaining. Fixed culture can be stored in PBS at 4 • C for up to four days.

Aliquoting and Freezing of BME Matrix
CRITICAL STEP: Frequent thawing of the matrix can lead to loss of polymerization efficiency. To avoid multiple freeze thaw cycles, the stock solution of BME matrix can be aliquoted into multiple tubes and stored at −80 °C. This procedure must be performed on the ice platform or any chilled surface. All tubes and pipette tips must be pre-chilled and placed on ice during aliquot preparation.
PAUSE STEP: All the aliquoted tubes must be stored at −80 °C immediately for future use.

Thawing of BME Matrix
CRITICAL STEP: BME matrix is thawed overnight in the 4 °C environment prior to starting a 3-D culture experiment. Because refrigeration temperatures may vary, the BME aliquots should be kept on ice in the refrigerator during the thawing process.
PAUSE STEP: Thawed BME polymerizes rapidly at temperatures above 15 °C. It is important to keep it on ice to prevent its solidification prior to coating culture vessels. Pre-chilled pipette tips and tubes must be used to prevent untimely gelling of BME during the coating procedure.

Embryonic Ventricular Cell Isolation
 Embryos (E11.5) from the timed-pregnant CD1 mice (Charles River Laboratories, Senneville, Quebec, QC, Canada) are taken out from the uterus by removing the placenta and kept in dishes containing PBS with 1× Ab/Am. The whole heart is removed from the embryo, and then the atria and outflow tracts are dissected out.


Both ventricles from each embryonic heart are placed in 0.2% v/v type I Collagenase in 1× PBS (~10 ventricles in 1 mL) and incubated for 45 min at 37 °C on a rocking platform and the ventricular tissue is digested.
CRITICAL STEP: Collagenase digestion of E11.5 ventricles should not exceed 45 min for optimal cell viability and recovery.
CRITICAL STEP: After 45 min of incubation, the tissue is triturated using a pipette tip for mechanical dissociation of the remaining tissue. Subsequently, dissociated CRITICAL STEP: Longer storage of fixed culture in PBS is not advisable as storage for more than four days causes loss of matrix integrity, and 3-D cell structure cannot be visualized. Immediate or next day processing yields a better outcome.

Immunofluorescence Staining of 3-D Culture
• Aspirate the PBS from the fixed 3-D culture chamber and wash the slide with PBSglycine at room temperature for 10 min.

Aliquoting and Freezing of BME Matrix
CRITICAL STEP: Frequent thawing of the matrix can lead to loss of polymerization efficiency. To avoid multiple freeze thaw cycles, the stock solution of BME matrix can be aliquoted into multiple tubes and stored at −80 °C. This procedure must be performed on the ice platform or any chilled surface. All tubes and pipette tips must be pre-chilled and placed on ice during aliquot preparation.
PAUSE STEP: All the aliquoted tubes must be stored at −80 °C immediately for future use.

Thawing of BME Matrix
CRITICAL STEP: BME matrix is thawed overnight in the 4 °C environment prior to starting a 3-D culture experiment. Because refrigeration temperatures may vary, the BME aliquots should be kept on ice in the refrigerator during the thawing process.
PAUSE STEP: Thawed BME polymerizes rapidly at temperatures above 15 °C. It is important to keep it on ice to prevent its solidification prior to coating culture vessels. Pre-chilled pipette tips and tubes must be used to prevent untimely gelling of BME during the coating procedure.

Embryonic Ventricular Cell Isolation
 Embryos (E11.5) from the timed-pregnant CD1 mice (Charles River Laboratories, Senneville, Quebec, QC, Canada) are taken out from the uterus by removing the placenta and kept in dishes containing PBS with 1× Ab/Am. The whole heart is removed from the embryo, and then the atria and outflow tracts are dissected out.  Both ventricles from each embryonic heart are placed in 0.2% v/v type I Collagenase in 1× PBS (~10 ventricles in 1 mL) and incubated for 45 min at 37 °C on a rocking platform and the ventricular tissue is digested.
CRITICAL STEP: Collagenase digestion of E11.5 ventricles should not exceed 45 min for optimal cell viability and recovery.
CRITICAL STEP: After 45 min of incubation, the tissue is triturated using a pipette tip for mechanical dissociation of the remaining tissue. Subsequently, dissociated tissue fragments are allowed to settle for 5 min for gravity separation of the fibrous tissue debris.
CRITICAL STEP: Cell extraction from 3D cultures could be for different purposes. For the sub-culture of cells, the cell extraction procedure should be gentler. Avoid forming a tight pellet. Cells are resuspended in culture media and can be plated to the fibronectin coated dish or slides. In our experience, the yield of extracted cells from 3-D matrix for subculture is very low, but a better result can be achieved by more intense optimization of cell extraction and subculture procedures.

Aliquoting and Freezing of BME Matrix
CRITICAL STEP: Frequent thawing of the matrix can lead to loss of polymerization efficiency. To avoid multiple freeze thaw cycles, the stock solution of BME matrix can be aliquoted into multiple tubes and stored at −80 °C. This procedure must be performed on the ice platform or any chilled surface. All tubes and pipette tips must be pre-chilled and placed on ice during aliquot preparation.
PAUSE STEP: All the aliquoted tubes must be stored at −80 °C immediately for future use.

Thawing of BME Matrix
CRITICAL STEP: BME matrix is thawed overnight in the 4 °C environment prior to starting a 3-D culture experiment. Because refrigeration temperatures may vary, the BME aliquots should be kept on ice in the refrigerator during the thawing process.
PAUSE STEP: Thawed BME polymerizes rapidly at temperatures above 15 °C. It is important to keep it on ice to prevent its solidification prior to coating culture vessels. Pre-chilled pipette tips and tubes must be used to prevent untimely gelling of BME during the coating procedure.

Embryonic Ventricular Cell Isolation
 Embryos (E11.5) from the timed-pregnant CD1 mice (Charles River Laboratories, Senneville, Quebec, QC, Canada) are taken out from the uterus by removing the placenta and kept in dishes containing PBS with 1× Ab/Am. The whole heart is removed from the embryo, and then the atria and outflow tracts are dissected out.  Both ventricles from each embryonic heart are placed in 0.2% v/v type I Collagenase in 1× PBS (~10 ventricles in 1 mL) and incubated for 45 min at 37 °C on a rocking platform and the ventricular tissue is digested.
CRITICAL STEP: Collagenase digestion of E11.5 ventricles should not exceed 45 min for optimal cell viability and recovery.
CRITICAL STEP: After 45 min of incubation, the tissue is triturated using a pipette tip for mechanical dissociation of the remaining tissue. Subsequently, dissociated tissue fragments are allowed to settle for 5 min for gravity separation of the fibrous tissue debris.


The supernatant is recovered, leaving debris in the tube, and the aqueous fraction containing cells is centrifuged at 1600 rcf (Eppendorf centrifuge, model#5415D) for 4 min at 4 °C. The supernatant is discarded, and the cell pellet is neutralized by two washes of culture media (DMEM containing 10% FBS and 1× Ab/Am).  Finally, cells are resuspended using 10% FBS-DMEM, and the cell number is determined using a hemocytometer. The cell suspension is resuspended in media to achieve desired cell density for 3-D cultures.
CRITICAL STEP: Prepare background wells for both 2-D and 3-D cell cultures with a similar number of cells. Background wells will have no primary antibodies and be added with secondary antibodies only. This helps in correction of raw values obtain by imaging system and avoid non-specific staining.

Aliquoting and Freezing of BME Matrix
CRITICAL STEP: Frequent thawing of the matrix can lead to loss of polymerization efficiency. To avoid multiple freeze thaw cycles, the stock solution of BME matrix can be aliquoted into multiple tubes and stored at −80 °C. This procedure must be performed on the ice platform or any chilled surface. All tubes and pipette tips must be pre-chilled and placed on ice during aliquot preparation.
PAUSE STEP: All the aliquoted tubes must be stored at −80 °C immediately for future use.

Thawing of BME Matrix
CRITICAL STEP: BME matrix is thawed overnight in the 4 °C environment prior to starting a 3-D culture experiment. Because refrigeration temperatures may vary, the BME aliquots should be kept on ice in the refrigerator during the thawing process.
PAUSE STEP: Thawed BME polymerizes rapidly at temperatures above 15 °C. It is important to keep it on ice to prevent its solidification prior to coating culture vessels. Pre-chilled pipette tips and tubes must be used to prevent untimely gelling of BME during the coating procedure.

Embryonic Ventricular Cell Isolation 
Embryos (E11.5) from the timed-pregnant CD1 mice (Charles River Laboratories, Senneville, Quebec, QC, Canada) are taken out from the uterus by removing the placenta and kept in dishes containing PBS with 1× Ab/Am. The whole heart is removed from the embryo, and then the atria and outflow tracts are dissected out.  Both ventricles from each embryonic heart are placed in 0.2% v/v type I Collagenase in 1× PBS (~10 ventricles in 1 mL) and incubated for 45 min at 37 °C on a rocking platform and the ventricular tissue is digested.
CRITICAL STEP: Collagenase digestion of E11.5 ventricles should not exceed 45 min for optimal cell viability and recovery.
CRITICAL STEP: After 45 min of incubation, the tissue is triturated using a pipette tip for mechanical dissociation of the remaining tissue. Subsequently, dissociated tissue fragments are allowed to settle for 5 min for gravity separation of the fibrous tissue debris.  The supernatant is recovered, leaving debris in the tube, and the aqueous fraction containing cells is centrifuged at 1600 rcf (Eppendorf centrifuge, model#5415D) for 4 min at 4 °C. The supernatant is discarded, and the cell pellet is neutralized by two CRITICAL STEP: Following in-cell westerns, stain the wells with Hoechst dye and determine the cellular DNA content using a fluorescence plate reader (BMG Labtech Polarstar Omega). Cellular content in each well can be used to normalize the respective in-cell western signals.

Statistical Analysis
Statistical analysis was performed using Graphpad Prism version 7 (Graphpad Software, San Diego, CA, USA). All data were compared using an unpaired Student t-test or the ANOVA with Tukey's multiple comparisons test and are presented as mean ± standard error of the mean (SEM). Differences of p < 0.05 were considered significant.

Results and Discussion
The 3-D cell culture methodologies provided an opportunity to observe the cell growth in a more physiologically relevant environment. For instance, normal 2-D cell cultures of neonatal or ES derived cardiomyocytes do not allow formation of elongated cardiac fibers under limited surface adherent conditions, whereas efficient cardiac fiber formation was reported in 3-D cell culture models [32]. In this study, we reasoned that 3-D cell culture conditions can improve embryonic ventricular cell differentiation into VCS cells. We tested three different types of BME matrices (Cultrex BME without reduced growth factors, Cultrex or Geltrex BME with reduced growth factors) and two different 3-D cell culture methods (cells on the matrix bed Vs. cells embedded in the matrix) for optimal embryonic ventricular cell differentiation studies. Cell seeding density has a major impact on the formation of spheroid cell bodies and connecting filamentous fiber formation in 3-D cultures [33]. Varying cell numbers (125,000-250,000 cells per chamber) were tested with a 70-100 µL matrix for optimal results. A lower cell number (<125,000 cells/chamber) resulted in a smaller size of spheroid cell bodies, and filamentous connections between them were rarely noticed. Cultures with a higher cell seeding densities (>250,000) led to the formation of giant cell clusters and revealed adverse effects on culture viability and beating quality over a 6-8-day culture period. Reduced nutrient diffusion to cells present in the core of the large clusters could be a possible reason for the reduced beating quality and shorter life of larger cell clusters as proposed in other studies [34]. Seeding of 125,000 to 250,000 E11.5 ventricular cells per chamber consistently resulted in the formation of spheroid cell bodies and connecting fibers with optimal viability and contractility.
Notably, the development of 3-D cell structures varied between the two cell cultures methods described here. Cells embedded in the matrix method consistently resulted in the formation of smaller spheroid cell bodies with limited inter-spheroid connections, cell contractility and viability ( Figure S1) when compared to the cells on top of the matrix bed method (Figure 1, Videos S1 and S2). Reduced mobility of cells within the matrix could be one of the potential reasons for cells not able to form larger clusters using the embedding method. Reduced viability of spheroid cell bodies in the embedding method was evident after 7 to 8 days culture by the loss of contractility and black discoloration. Whereas in the case of the cells-on-top method, robust and well synchronized contractions were evident even after 13-15 days of culture. As a result, the cells-on-the-matrix-bed method was opted for all subsequent experiments.
We observed uniform distribution of E11.5 ventricular cells throughout the BME matrix using cells over the matrix bed method (Section 3.3) immediately after plating (Day 0, Figure 1A). Organized cell clusters began to form by day 2 and subsequently contractile spheroid structures developed by day 4 in all three different types of BME matrices tested ( Figure 1B-D). Notably, cells grown on Cultrex BME matrix without reduced growth factors formed larger clusters at day 4 when compared to cells grown on either Cultrex or Geltrex BME matrix with reduced growth factors ( Figure 1C,D). The spheroid cell bodies started to develop fibrous outgrowths by day 6 and were subsequently connected either by thin or thick filamentous contractile structures by day 8 (Figure 1E-G). We observed the formation of thick filamentous connecting structures more frequently near the chamber's wall ( Figure 1G). At this point, we do not know the possible reasons for the higher attachment of filaments to the walls of culture vessels. In addition to inter-spheroid connecting fibers, the spheroid structures also developed thin fibrous outgrowths/extensions by day 8 in culture ( Figure 1H). Ventricular cells grown in BME matrices with reduced growth factors formed fewer fibrous outgrowths from spheroid structures and revealed less beating activity when compared to regular Cultrex BME (without reduced growth factors) after day 8 in culture ( Figure S2; also compare Video S2 and Video S3). As a result, the regular Cultrex BME matrix in combination with cells on the matrix bed method was routinely used for all subsequent experiments.
The effects of exogenous addition of ANP (1 µg/mL) and or an NPRA inhibitor A71915 (A7, 1 µM) on the growth and contractility of embryonic ventricular cells grown in 3-D cultures were determined (Figure 2A,B). The 3-D cultures were treated with these agents on alternate days for 8 days (see the timeline chart in Table 1). Previous studies demonstrated the critical role of ANP and its high affinity receptor NPRA in the embryonic ventricular cell proliferation and differentiation [11,14] as well as in cardiac hypertrophy and other disease states [35]. While ANP treatment significantly increased the mean area and the number of contractile spheroid cell bodies, co-treatment of ANP and A7 led to significant decreases in growth and contractility of spheroids cell bodies (Figure 2A,B). Furthermore, ANP treatment significantly increased the average number of fibers originating from the spheroid cell bodies with or without A7 co-treatment when compared to the control 3-D cultures ( Figure 2C).
Previous molecular modeling studies showed that both A7 and ANP can interact with some common and many distinct amino acid residues in the NPRA-extracellular binding domain (ECD) dimers and subsequently stabilize the ECDs in which the juxta-membrane regions are in the proximity of each other but in opposite orientations. These differential confirmations are critical for either stimulation or inhibition of the cytoplasmic guanylyl cyclase domains [36][37][38]. Moreover, A7 was shown to act as competitive inhibitor of the radiolabelled ANP binding to the NPRA receptor [38,39]. As the ligand and antagonist were added at the same time to 3D cultures in this study, it is plausible that the net receptor conformational changes induced by A7 by replacing the bound ANP could lead to more pronounced inhibitory effects and thus cause reductions in the spheroid size and contractility when compared to ANP or A7 effects alone. While the absence of inhibitory effects on mean fiber numbers seen with combined addition of A7 and ANP ( Figure 2C) may not be explained by the receptor conformational changes but could be explained by the existence of cGMP-independent NPRA signaling pathways [40]. Additional studies are required to identify molecular or biophysical mechanisms responsible for the differential effects observed with the combined addition of ANP and A7. A71915 (A7, 1 μM) on the growth and contractility of embryonic ventricular cell in 3-D cultures were determined (Figure 2A,B). The 3-D cultures were treated w agents on alternate days for 8 days (see the timeline chart in Table 1). Previous demonstrated the critical role of ANP and its high affinity receptor NPRA in the onic ventricular cell proliferation and differentiation [11,14] as well as in cardia trophy and other disease states [35]. While ANP treatment significantly incre mean area and the number of contractile spheroid cell bodies, co-treatment of A A7 led to significant decreases in growth and contractility of spheroids cell bodies 2A,B). Furthermore, ANP treatment significantly increased the average number originating from the spheroid cell bodies with or without A7 co-treatment wh pared to the control 3-D cultures ( Figure 2C).  Open circles represent individual data points for each group. * p < 0.05, ** p < 0.005,*** p < 0.0005 and **** p < 0.0001 for indicated group comparisons, One-way ANOVA with Tukey's post hoc test. We next examined the suitability of 3-D culture system for VCS cell differentiation studies. Several growth factors, signaling pathways and transcription factors have been shown to play a critical role in cardiac conduction system cell differentiation [41][42][43]. The gap junction protein Cx40 expression has been used as a valid marker for VCS cell differentiation studies [11,44,45]. Confocal imaging of fixed spheroid cell bodies revealed a robust Cx40 immunostaining within the cell bodies as well as within the connecting fibers after 8 days in culture ( Figure 3A-C). During cardiac morphogenesis, atrial specific gene expression (e.g., MLC-2a) was shown to be irreversibly downregulated in the developing ventricular myocardium [46]. In this study, cells were isolated from E11.5 ventricles after removing both atria and the outflow tracts. As a result, Cx40 expressing cells observed in this study represent exclusively ventricular derived conduction system cells and are unlikely to be derived from atrial cells.
x FOR PEER REVIEW   Figure 3D,E). Subsequently, an in-cell western detection technique was tested for marker protein expression levels in 2-D versus the 3-D cultures grown in microwell culture plates. In-cell western analysis also revealed significant increases in the expression of Cx40 and sarcomeric myosin protein levels in cells cultured under 3-D conditions compared to cells grown in 2-D cultures for 8 days (Figure 4, normalized for cellular DNA content). Figure 3. Analysis of Cx40 expression (ventricular conduction system (VCS) marker protein and 3-D cultures of E11.5 ventricular cells. (A) Representative merged confocal image of tures stained with Cx40 antibodies (green, arrows) and Hoechst nuclear dye, (B,C) The sa was imaged under brightfield (B) or Hoechst fluorescence alone (C). A-C: Arrows indicate ative location of Cx40 expressing cells, Bar = 50 μm, (D) Western blot analysis of Cx40 in me lysates extracted from three independent samples each from E11.5 ventricular cells culture (N = 3) and 3-D (N = 3) conditions for 8 days, Lower panel represents pan-cadherin (Cadh) sion levels which was used to normalize the relative levels of Cx40, (E) Signal intensities for shown in panel D were quantified using the acquisition software from Odyssey DLx (LI-C Cx40 expression levels relative to the pan-cadherin (Cadh) were shown. N = 3 independen tions for both 2-D and 3-D cultures. Each bar represents mean ± SEM. Open circles represe vidual data points for each group. * p < 0.05 for 2-D Vs. 3-D, Student's unpaired t-test.

Conclusions
In this paper, we have summarized detailed protocols for efficient differentiation of embryonic ventricular cells into VCS cells using 3-D culture methods. Our results suggest that 3-D cultures can generate a larger number of VCS cells and promote higher levels of myocyte maturation compared to 2-D cultures and are better suited to validate the effects of different agonists and antagonists to study developmental pathways.

Supplementary Materials:
The following supporting information can be downloaded at https://www. mdpi.com/article/10.3390/mps5030050/s1. Figure S1: Examples of cells embedded in the matrix (A) and cells on the top of the matrix cultured for 5 days in Cultrex BME. Note the absence of spheroid formation in cells embedded in the matrix method. Bar = 100 µm. Figure S2: (A) Ventricular cells grown in Geltrex BME matrix with reduced growth factors (Geltrex-redGF) revealed significantly lower beating frequencies when compared to cells grown in regular Cultrex BME (without reduced growth factors) after day 8 in culture, (B) Percentage of beating spheroid cell bodies did not differ significantly between cells grown in Geltred-redGF and Cultrex BME matrices, N = 5 independent experiments, each bar represents mean ± SEM. * p < 0.05, Student's unpaired t-test. Video S1: Examples of a contracting thin filament and spheroid cell bodies in E11.5 ventricular cells cultured on Cultrex BME using cells on top method. Video S2: Examples of a contracting thick filament and spheroid cell bodies in E11.5 ventricular cells cultured on Cultrex BME using cells on top method and Video S3: Limited spheroid cell body growth, contractility and connecting fiber formation in E11.5 ventricular cells grown on Geltrex BME with reduced growth factors.