Department of Biomedical Engineering, Tufts University
Ye, K. Y., Sullivan, K. E., Black, L. D. Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering . J. Vis. Exp. (55), e3251, doi:10.3791/3251 (2011).
Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.
1. Neonatal cardiomyocyte isolation - preparation (day before)
Solutions created in this section: PBS-Glucose solution, stop media.
2. Neonatal cardiomyocyte isolation - preparation (day of harvest)
Be sure to maintain sterility
Solutions used in this section: PBS-glucose solution, Betadine
3. Neonatal cardiomyocyte isolation - heart dissection
Solutions used in this section: Betadine, PBS-glucose solution
4. Neonatal cardiomyocyte isolation - myocyte isolation
Solutions created/used in this section: PBS-glucose solution, collagenase solution, stop solution
5. Casting fibrin gel constructs - preparation for creating fibrin gels (done well in advance)
Solutions created in this section: fibrinogen stock solution, thrombin stock solution, Pluronics solution, myocardial construct media.
6. Casting fibrin gel constructs - preparation for creating fibrin gels (right before making the fibrin gel constructs)
Solutions used in this section: Pluronics solution
7. Casting fibrin gel constructs via injection molding
Solutions created in this section: F solution, T solution, cell solution.
8. Analysis techniques (after 2 weeks in culture) - contraction force testing
Solutions used in this section: DMEM, myocardial construct media.
9. Analysis techniques (after 2 weeks in culture) - Live-Dead Assay for viability (with Invitrogen Live/Dead Assay)14:
Solutions used in this section: EthD-1 stock solution, calcein AM stock solution PBS
10. Analysis techniques (after 2 weeks in culture) - immunohistochemistry for important myocyte proteins:
Solutions used in this section: PBS, 4% paraformadehyde in PBS, 5% donkey serum in PBS, antibodies in PBS, 0.1 ng/mL Hoechst 33258 in PBS.
11. Representative results / Outcomes:
The cardiomyocyte fibrin construct initially covers the entire width of the mold (Figure 2B). No bubbles should exist in the construct and it should look uniform across the entire length. After two weeks of culturing, the construct contract to approximately 1/4 of the initial width (Figure 2C).
When the construct is electrically paced in our custom contraction force device (Figure 3A), twitch force data can be generated as shown in Figure 3B. The waveform can be analyzed separately in MATLAB (MathWorks) to determine the force, rate of contraction, and rate of relaxation. Twitch forces of approximately 1.3 mN are expected 6.
Cell viability of the construct is dependent on the depth of the construct, due to the diffusion limitations of oxygen into the construct. On the surface of the construct, Figure 4A, high cell viability is observed. With confocal microscopy, Figure 4B, the aligned structure of the construct is observed due to the Myosin Heavy Chain, MHC, is important for contraction, shown in red. Connexin 43, shown in green, is necessary for cellular coupling between myocytes.
Figure 1: Overview of the encapsulation process
Figure 2: A) Separate and combined mold parts for creating fibrin gels. From left to right: two Teflon washers, two silicone o-rings, a Teflon rod, a Teflon tube, a completed mandrel, the outer casing for the mandrel, and the plunger). B) Construct on mold immediately after ejection from the outer casing (day 0). C) Compacted construct on mold (red arrow), following 13 days of culture.
Figure 3: A) Custom contraction force measurement system for recording twitch force. A force transducer with a post measures the contraction force and outputs the results into a computer. A bath containing two carbon electrodes with wires connects to an electrical stimulator which paces the construct. The two posts hold the construct in place. B) Sample twitch force waveform data generated with electrical stimulation at 0.5 Hz.
Figure 4: A) Live/Dead assay of construct, day 13 (scale bar = 400 μm). Green represents the live cells and red represents the dead cells. B) Confocal Image of Myosin Heavy Chain (red), Connexin 43 (green) and Hoescht nuclear stain (blue) (scale bar = 10 μm).
|F solution||T solution||Cell Solution|
|Fibrinogen||112 μl||Thrombin||17 μl||Cells in DMEM||170 μl|
|Total||670 μl||Total||170 μl||Total||170 μl|
Table 1. Fibrin gel solutions, quantities for 1 mL of gel.
Note: Fibrinogen = 33 mg/mL Fibrinogen in 20 mM HEPES buffered Saline
HEPES = 20 mM HEPES buffered saline
Thrombin = 25 U/mL solution in 0.81% NaCl solution
Ca++ = 2 N Calcium Chloride solution
The encapsulation of neonatal rat cardiomyocytes in fibrin gels results in a consistent and viable three-dimensional in vitro model of the myocardial system. Fibrin is a preferred biomaterial because when the cells are entrapped, they are metabolically active and capable of compacting, remodeling and recreating an extracellular matrix that is consistent with native heart tissue 12. Because we allow the cardiomyocytes to align themselves in this environment, their functionality is more characteristic of cardiac muscle resulting in larger contraction force as compared to isotropic tissues 6. For potential therapeutic applications, it is necessary to encapsulate cells within a material that promotes both viability and functionality. The protocols presented here demonstrate an efficient and accurate means for creating a fibrin network to control cardiac cell behavior in a three dimensional microenvironment.
A few potential issues may arise during the creation and culture of these constructs. One potential issue is the maintenance of cell viability prior to encapsulation, which will significantly affect the functionality of the construct. Effort should be made to limit cell death following the isolation by reducing the time between the isolation and the encapsulation of the cells within the fibrin gel. Constructs should be provided media every other day on a strict schedule. In addition, it is important to ensure homogeneity in all of the solutions used. If the mixture of fibrinogen, thrombin and cells produces a heterogeneous environment, the ability of the cells to remodel the ECM, mechanically couple and contract is potentially obstructed. It is also important to prevent the formation of air bubbles during the injection of the constructs in order to prevent disturbances in the continuity of the engineered tissue. One way to alleviate this issue is draw more gel than is needed to make the construct into the syringe and inject slowly. Lastly, once the fibrin matrix has set and has been in culture medium for 24 hours, it is essential to detach the construct from the sides of the ring mold to promote the cell-based compaction of the fibrin gel. Keeping the construct in the middle of the mold facilitates gas and nutrient exchange. Adherence to the sides of the ring mold may also disrupt the desired cellular alignment.
It is important to note that conscious decapitation is used as the method of euthanization in this protocol, which is an acceptable method under the guidelines from both the National Institutes of Health and the American Veterinary Medical Association. However, some institutions recommend anesthetic use followed by decapitation for neonatal rats. We have chosen conscious decapitation because it ensures the minimal amount of time under hypoxic conditions for the excised heart tissue/cells. Relatively small amounts of hypoxia can lead to myocyte ischemia and potentially myocyte death, which could significantly affect the outcomes of this protocol.
No conflicts of interest declared.
This work was supported by the National Institutes of Health - National Heart, Lung and Blood Institute (Award # R00HL093358 to L.D.B.).
|Sodium phosphate dibasic||Sigma-Aldrich||S7907||PBS|
|Potassium phosphate monobasic||Sigma-Aldrich||P5655||PBS|
|hemostat||Fine Science Tools||91308-12||Isolation|
|fine tweezers||Fine Science Tools||11251-20||Isolation|
|large scissors||Fine Science Tools||91401-14||Isolation|
|micro-scissors||Fine Science Tools||91501-09||Isolation|
|scalpel handle||Fine Science Tools||10008-13||Isolation|
|scalpel blade||Fisher Scientific||08-918-5A||Isolation|
|Absorbent bench underpad||VWR international||56617-014||Isolation|
|sterile drape||Fisher Scientific||GM42526||Isolation|
|autoclave bag||Fisher Scientific||01-812-54||Isolation|
|gauze pad||Fisher Scientific||13-761-52||Isolation|
|betadine||Purdue Products L.P.||67618-150-01||Isolation|
|sterile gloves||Fisher Scientific||19-020||Isolation|
|sterile transfer pipette||Fisher Scientific||9962||Isolation|
|Teflon rod 1/4 inch diameter||McMaster-Carr||8546K11||Mold part|
|Teflon tube 1/4 inch ID, 1/2 inch OD||McMaster-Carr||8547K31||Mold part|
|Silicone O-Ring 1/4 inch ID, 1/2 inch OD||McMaster-Carr||9396K204||Mold part|
|Teflon tube 1/4 inch ID, 5/16 inch OD||McMaster-Carr||52355K14||Mold part|
|Kendall monoject syringes 6cc||Fisher Scientific||05-561-41||Mold part|
|BD syringe 3cc||Fisher Scientific||309585||Mold part|
|1 M HEPES||Sigma-Aldrich||H0887||Construct|
|0.2 micron filter||Fisher Scientific||SCGVT05RE||Construct|
|40 micron cell strainers||Fisher Scientific||22-363-547||Construct|
|0.45 micron bottle top filter||Corning||430627||Construct|
|glass pre-filter||EMD Millipore||AP2007500||Construct|
|18G 1 1/2 inch long needle||Fisher Scientific||14-826-5D||Construct|
|21G 1 inch needle||Fisher Scientific||14-826C||Construct|
|construct jars||Fisher Scientific||2116||Construct|
|Fetal bovine serum||Invitrogen||16000||Media|
|aminocaproic acid||Acros Organics||103305000||Media|
|Paraformaldehyde, 16%||Electron Microscopy Sciences||15710||Histology|
|Optimal cutting temperature (OCT)||Ted Pella, Inc.||27050||Histology|
|Mouse MYH1/2/4/6 primary antibody||Santa Cruz Biotechnology, Inc.||SC-32732||Histology|
|Rabbit Connexin 43 primary antibody||Cell Signaling Technology||3512||Histology|
|Dylight 549-conjugated donkey anti-mouse secondary antibody||Jackson ImmunoResearch||715-505-151||Histology|
|Dylight 488-conjugated Donke anti-rabbit secondary antibody||Jackson ImmunoResearch||711-485-152||Histology|