Intraspinal Cavity Injection of Human Mesenchymal Stem Cells and Tracking their Migration into the Rat Brain

Resumen

Several routes of administration can be used to deliver mesenchymal stem cells (MSCs) to the brain. In the present study, MSCs were delivered throughout the neuraxis and brain via intra-spinal cavity injection. MSCs were injected into the spinal cavities of rats, and stem cell migration was tracked and quantified.

Resumen

Mesenchymal stem cells (MSCs) have been studied for the treatment of various diseases. In neurodegenerative diseases involving defects in both the brain and the spinal cord, the route of administration is very important, because MSCs must migrate to both the brain and the spinal cord. This paper describes a method for administering MSCs into the spinal canal (intraspinal cavity injection) that can target the brain and spinal cord in a rat model. One million MSCs were injected into the spinal canals of rats at the level of lumbar vertebrae 2-3. After administration, the rats were euthanized at 0, 6, and 12 h post-injection. Optical imaging and quantitative real-time polymerase chain reaction (qPCR) were used to track the injected MSCs. The results of the present study demonstrated that MSCs administered via the spinal cavity could be detected subsequently in both the brain and spinal cord at 12 h. Intraspinal cavity injection has the advantage of not requiring general anesthesia and has few side effects. However, the drawback of the low migration rate of MSCs to the brain must be overcome.

Introducción

Mesenchymal stem cells
Under disease conditions, MSCs secrete disease-specific therapeutic substances via paracrine actions¹ that have been reported to regulate immune responses, restore damaged tissues, and remove toxic substances². Therefore, MSC therapy is considered more effective than single-target therapy in treating multifactorial diseases such as Alzheimer's disease and sarcopenia^3,4,5,6. Additionally, in contrast to pharmaceuticals, MSCs have a homing effect, moving to the region of the damaged tissue by recognizing inflammatory cytokines or chemokines in the body^7,8. Unfortunately, only a subset of the cells reach the damaged area, and the viability of MSCs decreases during migration^9,10,11,12. Thus, to maximize the therapeutic efficacy of MSCs, it is necessary to deliver viable cells to the target site. Therefore, when administering MSCs, it is important to choose the proper route of administration, based on the nature of the target disease.

Injection route
There are numerous routes by which therapeutic agents are administered to patients. The most common methods are intravenous injection into the systemic circulation, oral administration, and subcutaneous or intramuscular injection. In neurodegenerative diseases, the main obstacle in delivering therapeutic agents to the brain is the blood-brain barrier (BBB). The BBB protects the brain from external pathogens by means of tight junctions between blood vessels and the brain parenchyma^13,14. However, the BBB also paradoxically prevents therapeutic agents from entering the brain parenchyma. Therefore, passage through the BBB is the main hurdle in the development of brain disease therapies^15,16. Intracerebral injection is performed to overcome this drawback by injecting target substances directly into the brain through surgical operation^17,18,19. However, the side effects of surgical interventions should be considered, especially as the needle damages neuronal cells during the procedure.

Intraspinal cavity administration
Intrathecal administration-the administration of drugs into the spinal canal or subarachnoid space-delivers drugs to the brain or neuraxis through the cerebrospinal fluid (CSF) and is a viable alternative to intracerebral injection. Intrathecal injections can be subdivided according to the injection site: lateral ventricle, cisterna magna, and spinal cavity. All three routes allow drugs or cells to disperse throughout the CSF into the brain and spinal cord. Drug delivery to the brain may be more efficient in the case of intracerebroventricular and intra-cisterna magna injections because the agent is injected close to the brain. However, intraspinal cavity injection has the advantages of not requiring general anesthesia or surgery for inserting an intraventricular reservoir, being generally safe²⁰, and can be repeatedly performed if necessary.

The purpose of this study was to validate intraspinal cavity administration as a means of delivering MSCs to both the brain and spinal cord. First, the intraspinal cavity was established in a rat model. Next, MSCs were labeled with a lipophilic tracer, DiD (DiIC18(5); 1,1-dioctadecyl-3,3,3,3- tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt), to evaluate the efficiency of stem cell migration to the spinal cord and brain. Ex vivo optical imaging was performed to assess cell dispersion. This simple protocol can be performed without surgical intervention and may be used for the purpose of not only administering stem cells, but also pharmaceuticals, antibodies, contrast media, and other substances intended to be delivered to the spinal cord or brain.

Protocolo

NOTE: This study was approved by the Institutional Animal Care and Use Committee (Approval number: 20170125001, Date: January 25, 2017) of the Samsung Biomedical Research Institute (SBRI) at Samsung Medical Center. As an accredited facility of the Association for Assessment and Accreditation of Laboratory Animal Care International, the SBRI acts in accordance with the guidelines set forth by the Institute of Laboratory Animal Resources.

1. Preparation of human Wharton's jelly-derived MSCs

1. Cultivation of human Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs)
   1. Thaw a vial of human WJ-MSCs quickly in a 37 °C water bath. Transfer the WJ-MSCs to a 50 mL conical tube, and add growth medium at a volume at least 10 x that of the cells (v/v). Pipet up and down to suspend the cells.
   2. Centrifuge at 300 × g for 5 min. Carefully discard the supernatant, and resuspend the cells.
      NOTE: Be careful not to discard the cell pellet.
   3. Seed WJ-MSCs in T175 flasks at a density of 5,000-6,000 cells/cm². Incubate WJ-MSCs in a 37 °C CO₂ incubator. Change the growth medium every 72 h until WJ-MSCs reach 80-90% confluency.
      ​NOTE: Generally, it takes 3-4 days for the MSCs to reach 80-90% confluency.
2. Subcultivation of human WJ-MSCs
   1. Discard the growth medium, and wash the cells with 10 mL of phosphate-buffered saline (PBS). Remove the PBS, and add 5 mL of 0.25% trypsin-disodium ethylenediaminetetraacetic acid (EDTA) (see the Table of Materials). Incubate the cells at 37 °C in a CO₂ incubator for 3 min until the WJ-MSCs detach from the culture flask.
   2. Add 5 mL of growth medium containing 10% fetal bovine serum to neutralize the 0.25% trypsin-EDTA. Collect the cell mixture and transfer it to a 50 mL conical tube. Wash the cell culture flask with 10 mL of growth medium, and collect the cells in a 50 mL tube using a sterile serological pipet.
   3. Centrifuge the cell mixture at 300 × g for 5 min. Discard the supernatant, resuspend the cells in 10 mL of growth media, and count the number of WJ-MSCs.
      ​CAUTION: Be careful not to discard the cell pellet.
   4. Seed WJ-MSCs at a density of 4,000-6,000 cells/cm², depending on the experiment.
3. Labeling WJ-MSCs with DiD dye and the preparation of WJ-MSCs for intraspinal cavity injection
   NOTE: The DiD dye-labeling procedure was performed following manufacturer's instructions.
   1. Detach WJ-MSCs when they reach 80% confluency, using the procedure mentioned above. Suspend WJ-MSCs at a density of 1 × 10⁶/mL in phenol-red-free minimum essential medium (MEM) α without serum.
   2. Add 5 µL of DiD labeling solution per 1 mL of cell suspension; mix gently with pipetting.
   3. Incubate for 15 min at 37 °C; centrifuge the cell suspension at 300 × g for 5 min.
   4. Remove the supernatant, and resuspend the WJ-MSCs in phenol-red-free MEM α at a density of 1 × 10⁶/0.2 mL.

2. Intraspinal cavity injection of WJ-MSCs

1. Preparation for intraspinal cavity injection
   1. Anesthetize 6-week-old Sprague-Dawley rats with 5% isoflurane; then, maintain anesthesia with 2% isoflurane throughout the surgical procedure.
      NOTE: Optimize the anesthetic conditions before starting the experiment.
   2. Shave the surgical area using an electric shaver for small animals.
      ​NOTE: The electric shaver can be replaced with a manual razor and shaving gel.
   3. Disinfect the surgical area using povidone-iodine. Create a 3 cm incision in the skin with a surgical blade. Resect the remaining skin and muscle tissue using a surgical blade and scissors. Reveal the spinous processes at lumbar 2-3 (L2-3).
2. Injection of DiD-labeled WJ-MSCs via the intraspinal cavity
   1. Place the rat in a prone position. Flex the rat's spine appropriately to widen the distance between the adjacent spinous processes, using sufficient amounts of paper tissue or other materials that can aid in maintaining the appropriate position.
   2. Fill a 1 mL syringe with 0.2 mL of DiD-labeled WJ-MSCs. Place a 23 G syringe-needle combination vertically between the spinous processes of L2 and L3, and insert the needle until it touches the vertebral body.
   3. When the needle touches the vertebral body, retract it by approximately 0.5 cm, placing the tip of the needle in the spinal canal. Tilt the syringe, and place the tip of the needle such that it points toward the rostral direction. Inject WJ-MSCs into the spinal cavity over a 1 min period.
      ​NOTE: The speed of injection should be optimized in advance.
   4. After injection, completely remove the syringe from the spinal canal. Suture the incision, and then disinfect the surgical site using povidone-iodine.
3. Post-procedure treatment
   1. Stabilize and restrain the rat to prevent any movement, placing it upside-down at a 45° angle for 15 min, while it is still under anesthesia. After 15 min, discontinue anesthesia, and wait for the rat to rouse.

3. Evaluation of intraspinal cavity injection

1. Euthanasia of the rats and isolation of the brain and spinal cord at 0, 6, and 12 h post-injection
   1. Anesthetize the experimental animals with 5% isoflurane; maintain anesthesia with 2% isoflurane during PBS perfusion.
   2. Make an incision below the diaphragm using surgical scissors. Open the incision with forceps, and cut the rib cage rostrally to expose the heart.
   3. Make a small hole in the right atrium, and insert a butterfly needle into the left ventricle. Perfuse 100 mL of cold PBS into the left ventricle for 4-5 min, until the liver is cleared of blood.
   4. After perfusion, make a long incision on the backside from the head to the tail using a surgical blade along the longitudinal plane. Isolate the remaining brain and the whole spine using surgical scissors, forceps, and a bone cutter. Remove the remaining ribs, connected bones, and flesh.
2. Ex vivo DiD fluorescent optical imaging
   1. Place the isolated tissues in the chamber of the optical imaging device.
   2. Set the parameters as follows: emission, 700 nm; excitation, 605 nm; and exposure time, 2 seconds, as photons per second per centimeter squared per steradian (p/s/cm²/sr). Capture the optical images.
      NOTE: All images should be acquired with identical illumination settings (lamp voltage, filters, f/stop, field of view, and binning).
   3. Draw three rectangular regions of interest (ROIs) of equivalent size for the spinal cord and one circle ROI for the brain using the drawing tool. Measure the fluorescent intensities of the ROIs .
3. Extraction of genomic DNA (gDNA) from the spinal cord and brain tissue
   1. Remove the skull and spine carefully using surgical forceps, scissors, and a rongeur.
   2. Harvest the brain and spinal cord from the skull and spine. Cut the spinal cord into three pieces (cervical, thoracic, and lumbar).
      ​NOTE: The harvested tissues must be stored at -80 °C if they are not analyzed immediately.
   3. Grind the tissues using a pre-chilled mortar, pestle, and liquid nitrogen. Extract gDNA using commercial products, following the manufacturer's instructions.
4. Quantitative real-time polymerase chain reaction (qPCR)
   1. Quantify the amount of gDNA in each sample using a spectrophotometer.
   2. Perform qPCR using 10 ng of gDNA per sample and human Arthrobacter luteus (ALU) primers^12,21.
   3. Calculate the exact number of WJ-MSCs in the samples using the ΔΔC_T method²².

Resultados

To evaluate the efficacy of intraspinal cavity injection of MSCs, DiD-labeled MSCs were used in the present study. Before injecting MSCs into the spinal cavity, the labeling efficacy was assessed in vitro using optical imaging and fluorescence microscopy (Figure 1). After staining the MSCs with the DiD labeling reagent using the procedure described in protocol section 3.1, optical images were taken of the culture plates on which DiD-labeled MSCs were seeded (Figure 1A

Discusión

The optimal route of administration for treatment with MSCs should be chosen depending on the target disease, the patient's condition, and the type of drug to be delivered. In cell therapies, including MSC therapy, direct injection of stem cells into the brain or intrathecally via the CSF must be considered as the cells cannot pass through the BBB¹⁹. Intraspinal cavity injection is relatively non-invasive and does not cause neuronal damage in the brain, unlike intracerebroventricular injection...

Materiales

Name	Company	Catalog Number	Comments
0.25% Trypsin-EDTA	Gibco-invitrogen	25200114	Cell culture
Fetal bovine serum	biowest	S1520	Culture medium supplement
gentamicin	Gibco-invitrogen	15710-072	Culture medium supplement
Gentra Puregene Tissue Kit	QIAGEN	158689	gDNA isolation
MEM, no glutamine, no phenol red	Gibco	51200038	WJ-MSC fomulation for injection
Miminum Essential Medium alpha	Gibco-invitrogen	12571063	WJ-MSC culture medium
Power SYBR Green PCR Master Mix	Applied Biosystems	4368577	quantitative real time PCR reagent
QuantStudio 6 Flex Real-Time PCR System	Thermo fisher	4485694	quantitative real time PCR
trypan blue	Gibco	15250061	Injection
Vybrant DiD Cell-Labeling Solution	invitrogen	V22887	Stem cell labeling solution
Xenogen IVIS Spectrum system	Perkin Elmer	124262	Optical imaging device