Continuous and Real-Time In Vivo Autobioluminescent Imaging in a Mouse Model

Abstract

In vivo small animal bioluminescent imaging has become an indispensable technique for interrogating the localization, health, and functionality of implanted cells within the complex environment of a living organism. However, this task can be daunting for even the most experienced researchers because it requires multiple animal handling steps and produces differential output signal characteristics in response to a number of experimental design variables. The recent emergence of autobioluminescent cells, which autonomously and continuously produce bioluminescent output signals without external stimulation, has the potential to simplify this process, reduce variability by removing human-induced error, and improve animal welfare by reducing the number of required needlesticks per procedure. This protocol details the implantation and imaging of autobioluminescent cells within a mouse model to demonstrate how cells implanted from a single injection can be imaged repeatedly across any post-implantation timescale without the need for further human–animal interaction or signal activation steps. This approach provides a facile means to continuously monitor implanted cellular output signals in real-time for extended time periods.

1. Introduction

In vivo bioluminescent imaging relies upon the combination of a luciferase and its corresponding luciferin substrate to generate an optical signal. Firefly luciferase serves as the prototypical example, wherein a D-luciferin substrate reacts with ATP to form AMP, CO₂, and an oxyluciferin that releases energy in the form of photons. The inherent disadvantage of many in vivo bioluminescent imaging procedures is that the luciferin substrate must be added exogenously to initiate the bioluminescent reaction. In an animal model, this typically requires subcutaneous, intraperitoneal, or intravenous injections of the luciferin substrate prior to each desired bioluminescent measurement. The route and timing of substrate injection and its biodistribution within and clearance from the animal all impact light emission kinetics [1, 2], which influences experimental outcomes and study conclusions if not carefully optimized and rigorously controlled for. Furthermore, the prerequisite for repeated luciferin injections limits the amount of imaging data that can be obtained from each animal and adversely affects animal welfare.

To circumvent the need to exogenously inject animals with a chemical substrate, autobioluminescent imaging instead uses a synthetic luciferase (derived from the Photorhabdus luminescens lux CDABE operon) designed specifically to express in mammalian cells using endogenous substrate scavenging for luciferin generation [3, 4]. This eliminates exogenous substrate addition, enables the continuous and autonomous emission of bioluminescence under in vitro, ex vivo, and in vivo formats, and removes the associated potential introduction of error resulting from luciferin quality variation, inconsistencies in dosage, the biological effects of foreign chemical introduction, or changes to substrate clearance rate resulting from changes in age or health among subjects [5, 6]. When paired with whole-animal imaging systems like those manufactured by PerkinElmer, MI Labs, or Spectral Instruments Imaging, autobioluminescent imaging allows small animal models to be noninvasively imaged at any point over their lifetime. This approach can therefore provide longitudinal real-time surveillance of biological processes. The techniques for working with autobioluminescent cells differ little from that of conventional bioluminescent cells expressing firefly luciferase, apart from the elimination of the multiple luciferin injection steps. The following protocol provides the details necessary for performing autobioluminescent in vivo imaging in small animal research models.

2. Materials

2.1. Autobioluminescent Cell Line

1. Autobioluminescent HEK293 human kidney cell line.

2.2. Animal Subjects

1. Five-month-old, female nu/nu (nude) immunocompromised mouse.

2.3. Cell Culture Medium

1. Dulbecco’s modified Eagle’s medium (DMEM).

2. Fetal bovine serum (FBS): 10% solution.

3. Penicillin/Streptomycin (PenStrep): 1% solution.

4. G418: 100 μg/mL.

5. Dulbecco’s phosphate-buffered saline (DPBS).

2.4. Implantation

1. Dulbecco’s phosphate-buffered saline (DPBS).

2. Trypsin: 0.05% solution.

3. Trypan Blue.

4. Isoflurane–oxygen anesthesia induction chamber.

5. Hypodermic needle: 25-gauge.

6. Syringe: 1 mL.

2.5. Hardware

1. IVIS Lumina K preclinical benchtop imaging system (PerkinElmer).

2. Hemocytometer or automated cell counting device.

3. Benchtop centrifuge capable of holding 15 mL tubes.

2.6. Software

1. Living Image 4.5.2 software (PerkinElmer).

3. Methods

3.1. Preparation of Autobioluminescent Cells

This protocol uses an autobioluminescent HEK293 human kidney cell. This cell type is robust, easy to culture, fast growing, and maintains a strong autobioluminescent output signal under a variety of conditions. However, it is important to select an appropriate cell type to support the specific goals of your experiment. If an autobioluminescent version of your cell type of choice is not readily available, the cells can be made autobioluminescent as described in Note 1.

1. Using a tissue culture treated T₇₅ flask, culture the autobioluminescent HEK293 cell line in 15 mL of DMEM supplemented with 10% FBS, 1% PenStrep, and 100 μg/mL G418 (see Notes 2 and 3). A T₇₅ culture flask should be used to ensure a sufficient number of cells are available for injection (see Note 4).

2. Incubate the cells at 37 °C in a humidified, 5% CO₂ incubator and refresh the medium every 2–3 days until the cells reach ~80% confluency.

3. When refreshing medium, the spent culture medium should be aspirated, the cells should be washed once with DPBS, then 15 mL of fresh culture medium should be added.

4. Upon reaching 80% confluence, the cells are ready to be harvested and prepared for implantation as described below.

3.2. Implantation of Autobioluminescent Cells into the Mouse Subject

This protocol details the subcutaneous implantation of autobioluminescent cells into the dorsal flank of an immunocompromised mouse subject. However, alternative injection sites such as tail vein, intraperitoneal, or intraorganellar; or an alternative subject type can be used based on the ultimate objective of the study (see Note 5). All animal studies should be conducted in compliance with protocols approved by an appropriate institutional animal care and use committee and the highest ethical standards should be employed. This protocol was conducted in accordance and compliance with all relevant regulatory and institutional agencies, regulations and guidelines.

1. Harvest the prepared autobioluminescent cells by aspirating the culture medium, washing once with 5 mL of 1× DPBS, and disassociating with 3 mL of 0.05% trypsin.

2. Incubate the cells in trypsin for ~2 min, or until they have detached, at 37 °C in a humidified, 5% CO₂ incubator.

3. Once the cells have detached, recover them into a 15 mL tube by adding 7 mL of DMEM supplemented with 10% FBS and 1% PenStrep to bring the total volume to 10 mL and transferring the full volume to the 15 mL tube.

4. Count the cells using a hemocytometer or automated cell counting system (see Note 6).

5. Concurrent with the cell counting procedure, centrifuge the remainder of the harvested cells at 250 × g for 5 min.

6. Aspirate the supernatant from the centrifuged cells and resuspend at a concentration of 1 × 10⁷ cells/mL in DPBS (see Note 7).

7. Prior to implantation, anesthetize the mouse subject to be assayed by placing it into an anesthesia induction chamber and supplying 5% (v/v) inhaled isoflurane in 1 L/min of oxygen. Closely monitor the status of the subject inside the induction chamber. The isoflurane level may need to be adjusted accordingly to achieve desired anesthesia level. Be careful to not overanesthetize the subject.

8. Using a 25-gauge needle and a 1 mL syringe, draw up the resuspended cells from step 6 into the syringe and inject 100 μL subcutaneously into the dorsal flank (see Note 8).

3.3. Imaging Autobioluminescent Output Signals

This protocol details the imaging of a single mouse subject in the IVIS Lumina K imaging system over a period of 30 min. Depending on the brand or model of imaging equipment being used, it may be possible to image multiple subjects simultaneously. Fortunately, if this is the case the same steps can be performed in parallel on each individual subject. Alternatively, the steps can be iterated across multiple subjects in series to achieve the same result.

1. Using the Living Image software, initialize the IVIS Lumina K and set the stage temperature to 37 °C.

2. Manually adjust the in-chamber anesthesia system to deliver 1.5% (v/v) inhaled isoflurane in 1 L/min of oxygen (see Note 9).

3. Place the anesthetized subject harboring the injected autobioluminescent cells into the imaging chamber of the IVIS Lumina K. Be sure to position the subject so that the area harboring the injected cells is facing toward the camera and so that the subject’s nose fits within the nose cone. Plug any unused nose cones with rubber stoppers to prevent the loss of anesthesia.

4. Close the door to the imaging system and ensure the interior lights are turned off.

5. Set the stage height setting to field of view C (see Note 10) so that the subject is centered in the field of view with minimal superfluous area visible.

6. Set the subject height to 1.50 cm and choose “use subject height” for focus.

7. Acquire a photograph by selecting the “Photograph” setting from the list of illumination settings and clicking the “Acquire” button. This photograph will show the position of the subject and allow you to determine if any changes in positioning need to be made prior to autobioluminescent signal acquisition. If repositioning is required, this step should be repeated to ensure the proper orientation has been achieved to provide an appropriate final image.

8. Once the subject has been appropriately positioned, select the “Luminescent” setting (both the “Photograph” and “Luminescent” settings should now be checked) from the list of illumination settings and input the following imaging acquisition settings (Fig. 1, see Note 11):

   Exposure Time: 10 min.

   Binning: Medium.

   F Stop: 1.

   Emission Filter: Open.

9. After confirming the settings are correct, click the “Sequence Set Up” button to open the sequence editor dialog.

10. Click the “Add” button to import your acquisition settings into the sequence editor dialog.

11. Check the “Number of Segments” box and adjust the corresponding number to 3. This will take three 10 min exposure images for a total of 30 continuous minutes of observation (Fig. 2).

12. After the imaging process completes, it can be repeated to obtain further data, or the subject can be returned to its cage and monitored until fully recovered from anesthetic. This recovery usually occurs within 5 min (see Note 12).

Fig. 1.

An example of the image acquisition settings for capture of autobioluminescent signal from subcutaneously implanted cells. Some of these settings, such as the binning, can be adjusted following image acquisition, but most cannot. It is important to ensure that the Photograph and Overlay checkboxes are selected in addition to the Luminescent checkbox when acquiring bioluminescent output signals. Depending on the autobioluminescent cell type used, the number of cells implanted, and the health of the implanted cells, the exposure time can be reduced down to as little as 1 s while still acquiring sufficient flux for pseudocolor localization

Fig. 2.

Example of an image capture sequence that will interrogate the subject continuously for half an hour using three 10-min exposures. The Add button and the check box for adjusting the number of segments in the sequence are circled

3.4. Processing Autobioluminescent Output Data

This protocol details the interrogation of a single implantation location from a single subject at one time point. The steps listed in this section can be duplicated for additional regions of interest, subjects, or time points as needed.

1. Double click the first image in the series to open that image for processing (Fig. 3).

2. Open the “ROI Tools” section of the Tool Palette in the Living Image software.

3. Select the circular ROI tool from the menu and use the cursor to draw a circle around the region of interest (ROI) in the pseudocolor image (Fig. 4, see Notes 13 and 14).

4. After the ROI has been drawn, click the “Measure ROI’s” button and a dialog box will open displaying the output signal level within the ROI (Fig. 5, see Note 15).

5. Click the “Select All” button to select the displayed data.

6. Click the “Copy” button to copy the information to the clipboard.

7. Paste the information into Microsoft Excel, or your preferred program for data analysis and storage.

8. Amend the file with any pertinent metadata that was not available for direct import from Living Image, save the file, and proceed with data analysis as appropriate.

Fig. 3.

Example of a pseudocolor image showing the location of subcutaneously implanted autobioluminescent cells within a nude mouse model. The location of the injected cells should be easily visible from the autobioluminescent signal

Fig. 4.

A region of interest (ROI) drawn around the location of the implanted autobioluminescent cells allows total signal output to be quantified

Fig. 5.

An example of the ROI measurement dialog box. These data show the quantification of autobioluminescent signal from within the ROI and can be used to make comparisons between different locations, time points, or treatments. Note the Configure button in the bottom left of the box. This will allow you to select additional metadata beyond the minimal default data (shown)