Name: Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT) (Video) | JoVE Journal
Uploaded: 2011-06-01T12:00:00.000+00:00
Duration: 7 min 33 s
Description: Learn in vivo bioluminescence imaging to monitor immune responses after engineered heart tissue implantation in rats. This non-invasive method enables longitudinal assessment of graft survival and rejection.

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5 minutes

Sonja Schrepfer, Lenard Conradi and Arne Hansen received funding from the Deutsche Forschungsgemeinschaft (DFG; SCHR992/3-1, SCHR992/4-1, CO 858/1-1, HA 3423/3-1). The work was also supported by grants from the European Union to Thomas Eschenhagen (EUGeneHeart and Angioscaff).

1. EHT Generation and Culture Conditions
Manufacture of fibrin-based EHT was performed as described elsewhere 4. In brief, to generate fibrin-based EHT for implantation purposes, a master mix was prepared containing cells isolated from neonatal ROSA/luciferase-LEW transgenic rat hearts, bovine fibrinogen and Matrigel. Agarose casting molds were prepared using Teflon spacers placed in 6-well culture dishes. After solidification of agarose, spacers were removed and silicone post racks were placed on culture dishes with posts reaching into the molds. To generate EHT, the mastermix was briefly mixed with a calculated amount of thrombin and pipetted into the mold. After polymerisation of fibrinogen, silicone racks with EHTs adhering to the posts were gently removed from the agarose casting molds, transferred to new 6-well cell culture dishes and maintained in a 37°C, 7% CO2 cell culture incubator using custom-made cell culture medium.
EHT were kept under cell culture conditions until day ten. During this time span, neonatal rat heart cells began to elongated, interconnect and align along force lines in between silicone posts. At the time of implantation, spontaneous coherent contractions were observed, deflecting silicone posts.
2. EHT Implantation
EHT implantation was performed in either immunocompetent Brown Norway (BN) rats, or immunodeficient nude rats to investigate the allogeneic immune response. BN and nude rats weighing 100 - 150 g were purchased from Charles River Germany (Sandhofer Weg 7, D-97633 Sulzfeld) and housed under conventional conditions, fed standard rat chow and water ad libidum. Immunodeficient rats are housed in sterile bedding, cages, and sterilized water is used. The German ethical review committee reviewed and approved the animal procedures. All surgical instruments are sterilized prior to use.
The implantation procedure are performed as follows:
Rat are anesthesized with isoflurane (2.5-3%) using an induction chamber. Human exposure to isoflurane can be reduced by working in a downdraft table or non-recirculating hood. Body temperature is maintained during the surgical procedure.
<ol>
	<li>Shave abdominal area and apply eye ointment to prevent the eyes from drying during anesthesia.</li>
	<li>Place the rat on its back and place a facemask over its nose and mouth to keep up the anesthesia.</li>
	<li>Disinfect the abdominal area using Betadine and then 80% ethanol. Swab from clean to dirty, from center of shaved area moving outward toward the haired area.</li>
	<li>Check reflexes pinching the hind feet to be sure that the rat is sufficiently anesthetized.</li>
	<li>Drape the animal and perform a midline abdominal incision separating the skin and muscle in two steps to open the abdomen. Hot bead sterilization is used between skin and muscle opening.</li>
	<li>Place the intestines in a warmed saline moisturized powder free glove. Fold the glove around the intestines to prevent loss of moisture.</li>
	<li>Remove fatty tissue and expose the spleen.</li>
	<li>Visualize and spread greater omentum with forceps.</li>
	<li>Carefully cut EHT from suspending silicone posts and transfer onto the greater omentum.</li>
	<li>Wrap the greater omentus around the EHT and fix using two single 7-0 prolene sutures (Ethicon, Norderstedt, Germany)</li>
	<li>Next move the intestines back into the abdomen.</li>
	<li>Flush the abdomen with prewarmed sterile saline.</li>
	<li>Close the muscle layer of the abdominal wall using 6-0 prolene running sutures (Ethicon, Norderstedt, Germany).</li>
	<li>Use 5-0 prolene (Ethicon, Norderstedt, Germany) running sutures to close the skin.</li>
	<li>While the rat is still in anesthesia, inject 5 mg/kg Carprofen subcutaneously.</li>
	<li>To provide sufficient analgesia for this type of procedure, an analgesic (such as Metamizol) is added to the drinking water (50 mg Metamizol per 100 ml) for pain medication for 3 days post transplantation.</li>
</ol>
3. Bioluminescence Imaging of EHT Following Implantation
The IVIS bioimaging platform (Xenogen, Alameda, CA, USA) is used for non-invasive bioluminescence imaging in-vivo and results are analyzed using the IVIS Living Image (Xenogen) software package. Imaging is performed daily starting 2 hours postoperatively.
Anesthetize rat with isoflurane (2.5 - 3%) using an induction chamber.
<ol>
	<li>Disinfect the abdominal and thoracic area widely using Provo-Iodine, next use 80% ethanol, repeat this step three times.</li>
	<li>Inject d-Luciferin (Xenogen; 375 mg/kg body weight) i.p.</li>
	<li>Place anaesthetized rats into the IVIS imaging chamber (up to 4 rats can be analysed at the same time).</li>
	<li>Assess signal intensity of luc+ cells inside EHT in units of photons/sec/cm2/steridianin the region of interest (ROI) at baseline (120 minutes after implantation) and at days 1, 2, 3, 5, 7, 10, and at weeks 2, 3 and 4. Note the peak signal (appears around 20 minutes after d-Luciferin injection).</li>
</ol>
Create a graph (Excel) showing the signal intensity over time.
4. Correlation of BLI Results and ELISPOT
The cellular in-vivo immune responses in immunocompetent BN and immunodeficient nude rats were studied after EHT transplantation. The spleen of recipient animals was harvested 5 days after EHT transplantation to isolate recipient splenocytes. Elispot assays using mitomycin-inhibited EHTs (Sigma Aldrich, St. Louis, MO, USA) as stimulator and 5 x106 recipient splenocytes as responder cells were performed according to the manufacturer's protocol (BD Biosciences) using IFN-γ and IL-4 coated plates to investigate TH1-, and TH2 response, respectively. IFN-γ- and IL-4 spots were significantly higher in the immunocompetent, model compared to the immunodeficient model (IFN-γ: p= 0.001; IL-4: p=0.023; Student's t-test).
5. Representative Results:
<img src="/files/ftp_upload/2605/2605fig1.jpg" alt="ELISpot diagram and photon decay graph after EHT transplantation." data-altupdatedat="1761809329000" data-alt="Figure 1"> 
Figure 1. Correlation of BLI results and ELISPOT a) The cellular in vivo immune responses in immunocompetent Brown Norway and immunodeficient nude rats were studied after EHT implantation. Recipient splenocytes were harvested 5 days after EHT implantation. IFN? and IL-4 expression was evaluated by ELISpot assays using mitomycin-inhibited EHT as stimulators and 5x106 recipient splenocytes as responder cells. Th1 and Th2 responses were significantly higher in the immunocompetent model compared to the immunodeficient model as evidenced by IFNγ- and IL-4 production respectively. b) Luc+ EHTs were transplanted into the greater omentus of either immunocompetent BN rats or immunodeficient nude rats. Cell survival was longitudinally followed by BLI. The rate of animals that rejected the transplanted EHTs is presented. All BN rats rapidly rejected the EHT transplants, whereas the cells survived in T cell deficient rats.

<ol><li>Eschenhagen, T., Zimmermann, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineering+myocardial+tissue%5BTitle%5D)+AND+%22Circ+Res%22%5BJournal%5D)">Engineering myocardial tissue.</a> Circ Res. 97, 1220-1231 (2005).</li>
<li>van der Bogt, K. E., Schrepfer, S., Yu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+transplantation+of+adipose+tissue-+and+bone+marrow-derived+mesenchymal+stem+cells+in+the+infarcted+heart%5BTitle%5D)+AND+%22Transplantation%22%5BJournal%5D)">Comparison of transplantation of adipose tissue- and bone marrow-derived mesenchymal stem cells in the infarcted heart.</a> Transplantation. 87, 642-652 (2009).</li>
<li>Hakamata, Y., Murakami, T., Kobayashi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((%22Firefly+rats%22+as+an+organ%2Fcellular+source+for+long-term+in+vivo+bioluminescent+imaging%5BTitle%5D)+AND+%22Transplantation%22%5BJournal%5D)">"Firefly rats" as an organ/cellular source for long-term in vivo bioluminescent imaging.</a> Transplantation. 81, 1179-1184 (2006).</li>
<li>Hansen, A., Eder, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+Drug+Screening+Platform+Based+on+Engineered+Heart+Tissue%5BTitle%5D)+AND+%22Circ+Res%22%5BJournal%5D)">Development of a Drug Screening Platform Based on Engineered Heart Tissue.</a> Circ Res. , (2010).</li>
</ol>

No conflicts of interest declared.

Before clinical implementation of EHT technology for cardiac repair, fundamental questions such as immunogenicity of both cellular and matrix components or graft survival after implantation need to be assessed in experimental models. In-vivo bioluminescence imaging represents a useful new technique for monitoring cell survival after transplantation. We successfully generated fibrin-based EHT containing luciferase positive (luc+) heart cells by adaptation of an established protocol 4. These EHT were im...

<table><tbody><tr><td colspan="4">&lt;ul&gt;&lt;li&gt;LEW-Tg(Gt(ROSA)26Sor-luc)11Jmsk rats (NBPR rat number 0299, http://www.anim.med.kyoto-u.ac.jp/NBR/)&lt;/li&gt;&lt;li&gt;Bovine fibrinogen (stock solution: 200 mg/ml plus aprotinin 5 &amp;#x03BC;g/ml fibrinogen in NaCl 0.9%, Sigma F4753)&lt;/li&gt;&lt;li&gt;Matrigel (BD Bioscience 356235)&lt;/li&gt;&lt;li&gt;DMEM (Biochrome F0415)&lt;/li&gt;&lt;li&gt;Horse serum (Gibco 26050)&lt;/li&gt;&lt;li&gt;Penicillin/streptomycin (Gibco 15140)&lt;/li&gt;&lt;li&gt;Insulin (Sigma-Aldrich I9278)&lt;/li&gt;&lt;li&gt;Aprotinin (Sigma Aldrich A1153)&lt;/li&gt;&lt;li&gt;d-Luciferin (Biosynth L-8220)&lt;/li&gt;&lt;li&gt;Enzyme-linked immunosorbent spot (ELISpot) assay plates (Hassa Laborbedarf, S2EM004M99)&lt;/li&gt;&lt;/ul&gt;</td></tr></tbody></table>

bioluminescence imaging for assessment of immune responses following implantation of engineered heart tissue (eht)

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or bioartificial scaffold materials, entrapment of cardiac myocytes in hydrogels such as fibrin or collagen and stacking of myocyte monolayers 1. These concepts aim at restoration of compromised cardiac function (e.g. after myocardial infarction) or as experimental models (e.g. predictive toxicology and substance screening or disease modelling). Precise monitoring of cell survival after implantation of engineered heart tissue (EHT) has now become possible using in-vivo bioluminescence imaging (BLI) techniques 2. Here we describe the generation of fibrin-based EHT from a transgenic rat strain with ubiquitous expression of firefly luciferase (ROSA/luciferase-LEW Tg; 3). Implantation is performed into the greater omentum of different rat strains to assess immune responses of the recipient organism following EHT implantation. Comparison of results generated by BLI and the Enzyme Linked Immuno Spot Technique (ELISPOT) confirm the usability of BLI for the assessment of immune responses.

This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or ...

The overall goal of the following experiment is to use in vivo bioluminescence imaging to study immune responses after implantation of engineered heart tissue or EHT in rats. This is achieved by first generating luciferase positive ehs. Next, EHS that stably express luciferase are transplanted into the greater omentum of recipient rats.Then in vivo bioluminescence imaging can be performed on recipient animals to monitor noninvasive longitudinal EHT survival and rejection. Using this model, the intensity and kinetics of acute rejection, for example, can be correlated with the decline of the observed bioluminescence signal intensity over time. The main advantage of this technique over existing methods like histology is that daily measurements allow for non-invasive longitudinal assessment of graft survival.This method can provide insight into the survival and rejection of engineered heart tissues, but it can also be applied to other questions. For example, metabolism of ehs. The bili technique will be demonstrated by Christiana Parman who's performing this technique on a daily basis.In my lab, transgenic heart cells are first isolated from Luciferase Lu neonatal rats to generate fibrin based DHT for implantation purposes, prepare a master mix containing cells isolated from neonatal luciferase, lu transgenic, rat hearts, bovine fibrinogen, and matrigel to X-D-M-E-M and NKM. Prepare aros casting molds by adding nine milliliters of 2%aros in PBS to each well in six well culture dishes, and then placing Teflon spacers into the wells. After the aros solidifies, remove the spacers and place silicone post racks on the culture dishes with the post reaching into the molds.To generate EHT, gently mix the master mix, then add a calculated amount of thrombin into the master mix and briefly mix. Then quickly pipette 1100 microliters of master mix into each mold. After the fibrinogen polymerizes, gently remove the racks with the posts that now have adherent EHT from the agros casting molds and transfer the racks to new six well cell culture dishes containing eight milliliters of custom made medium per well.EHT are maintained for 10 days in a 37 degree Celsius, 7%carbon dioxide cell culture incubator. And the medium is changed three times a week. During this culture period, neonatal rat heart cells will begin to elongate, interconnect, and align along force lines in between silicone posts.At the time of implantation, spontaneous coherent contractions will be observed. Sterilize the instruments for surgery by autoclave prior to use warm 30 milliliters of sterile saline solution. Rats for implantation should weigh 100 to 150 grams and be housed under conventional conditions provided with standard rat chow and water ad libido.In this video, brown Norway rats are used after anesthetization, sedate the rats prior to surgery and confirm sedation by lack of response to toe pinch while the rat is under anesthesia, inject five milligrams per kilogram of body weight, carens subcutaneously into the plank, shave the abdominal area and apply eye ointment to prevent the animal's eyes from drying out. During anesthesia, disinfect the abdominal area using Betadine, and then 80%ethanol swabbing from clean to dirty. Repeat this disinfection.Step three times, drape the animal and perform a midline abdominal incision, separating the skin and muscle in two steps to open the abdomen. Use hot bead sterilization on tools between cuts. Dampen a powder freak glove with the saline.Place the intestines inside the glove. Fold the glove around the intestines to prevent moisture loss. Spread the incision and expose the spleen.Identify and spread out the greater momentum with forceps. Carefully cut the EHT from the suspending silicone posts and transfer it onto the greater momentum. Wrap the greater momentum around the EHT and fix it closed using two single seven oh proline sutures.Next, move the intestines back into the abdomen. Flush the abdomen with one milliliter of prewarm sterile saline. Close the muscle layer of the abdominal wall using six oh proline running sutures.Use five oh proline running sutures to close the skin to provide sufficient analgesia for this type of procedure. At 50 milligrams per 100 milliliters of ole to the drinking water for three days post implantation. The IVUS Bioimaging platform is used for non-invasive bioluminescence Imaging in vivo and results are analyzed using the IVUS living image software.Package imaging is performed daily, starting two hours postoperatively. Prior to visualizing the EHT bioluminescence, anesthetize the rat and confirm sedation by toe. Pinch disinfect the abdominal area.Inject Lucifer at 375 milligrams per kilogram of body weight intitally and place the anesthetized rat into the ivu imaging chamber. Up to four rats can be analyzed at the same time. Use the IVUS living image software to observe and record the peak signal, which appears approximately 20 minutes after Lucifer injection.Then establish the baseline signal intensity of luciferase positive cells inside the EHT in units of photons per second per centimeter squared per Ceridian in the region of interest, 120 minutes after implantation and at days 1, 2, 3, 5, 7, 10, and weeks two, three, and four. The data can be expressed in graph form, showing the signal intensity over time. The cellular in vivo immune responses in Immunocompetent Brown, Norway and Immunodeficient.Nude rats were studied after EHT implantation recipient SP cytes were harvested five days after EHT implantation. Interferon, gamma and IL four expression were evaluated by lispa assays using mitomycin inhibited EHT as stimulators and five times 10 of the six recipient cytes as responder cells. TH one and TH two responses were significantly higher in the immunocompetent model compared to the Immunodeficient model as evidenced by interferon, Gemma and IL four production respectively.LUCIFERASE positive EHS were transplanted into the greater omentum of either immunocompetent PN rats or immunodeficient nude rats. Cell survival was longitudinally followed by BLI. The rate of animals that rejected the transplanted EHS is presented.All BN rats rapidly rejected the EHT transplants, whereas the cell survived in T-cell deficient Rats Once mastered surgeries can be done within minutes if the technique is performed properly after surgery. Other methods like immunological essays and histology can be performed in order to answer other questions regarding cell infiltration and humeral response.

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Learn in vivo bioluminescence imaging to monitor immune responses after engineered heart tissue implantation in rats. This non-invasive method enables longitudinal assessment of graft survival and rejection.

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

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